Bismuth-thiol compositions and methods of use

ABSTRACT

The invention relates to Bis-thiol compounds and pharmaceutical preparations thereof. The invention further relates to methods of treating, managing or lessening the severity of pulmonary infections in a subject, the method comprising administering to the subject a bismuth-thiol (BT) composition that comprises at least one BT compound.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.16/528,145, filed Jul. 31, 2019, which claims the benefit of U.S.Provisional Application No. 62/712,563, filed Jul. 31, 2018 and U.S.Provisional Application No. 62/800,925, filed Feb. 4, 2019, the contentsof which are incorporated herein by reference in their entireties.

BACKGROUND

Cystic fibrosis (CF) manifests as a clinical syndrome characterized bychronic pulmonary infection as well as by gastrointestinal, nutritional,and other abnormalities. The genetic basis for CF is awell-characterized, severe monogenic recessive disorder that arises frommutations in the cystic fibrosis transmembrane conductance regulator(CFTR) gene. Patients often have chronic pulmonary infections, such aswith Pseudomonas aeruginosa. Ultimately, 80 to 95% of patients with CFsuccumb to respiratory failure brought on by chronic bacterial infectionand concomitant airway inflammation.

Lungs of CF patients are often colonized or infected in infancy andearly childhood with organisms, such as Staphylococcus aureus andHaemophilus influenzae, that may damage the epithelial surfaces, leadingto increased attachment of, and eventual replacement by, P. aeruginosa.Chronic infection with P. aeruginosa is the main proven perpetrator oflung function decline and ultimate mortality in CF patients. Chronic P.aeruginosa infection leads to epithelial surface damage and airwayplugging, progressively impairing airway conductance, which results in adecline in pulmonary function. P. aeruginosa also develops resistance tomany common antibiotics which makes eradication of the infection quitedifficult. One factor in developing resistance is the tendency of P.aeruginosa and other CF associated lung pathogens to form biofilmstendency to create biofilms that are more difficult for antibiotics topenetrate. In addition, the prevalent use of antibiotics in treatinginfections in general has led to multi-drug resistant (MDR) strains ofbacteria, such as P. aeruginosa, and Staphylococcus aureus.

Long-term inhaled antibiotic therapy is now standard of care for chronicmaintenance treatment in stable patients. Effective antibioticconcentrations can be achieved in the airways by nebulization, avoidingside effects of intravenous antibiotics. Colistimethate sodium,amikacin, aztreonam and tobramycin have been administered to patients byinhalation. However, there is an unmet need for treatment of pulmonarymicrobial (e.g. bacterial or fungal) infections in CF patients that isbroad spectrum, durable and potent against even the most resistant ofmicrobial (e.g. bacterial or fungal) species.

SUMMARY

Disclosed herein are methods of treating, managing or lessening theseverity of cystic fibrosis (CF) symptoms and infections in a subject,the method comprising administering to the subject a bismuth-thiol (BT)composition that comprises at least one BT compound.

In certain embodiments, the present disclosure provides a pharmaceuticalcomposition suitable for use in a subject for treating, managing orlessening the severity of cystic fibrosis (CF) symptoms and infections,comprising an effective amount of any of the compounds described herein(e.g., a compound of the disclosure, such as a bismuth-thiol compound,or a pharmaceutically acceptable salt thereof), and one or morepharmaceutically acceptable excipients.

In certain embodiments, the present disclosure provides an aerosolcomprising a plurality of dispersed liquid droplets in a gas, saidliquid droplets comprising a BT composition comprising at least one BTcompound suspended therein, wherein the BT compound comprises bismuthand/or a bismuth salt and a thiol-containing compound; and wherein atleast 60%, 65%, 70, 75%, 80%, 90%, or 95% of the liquid droplets have amass median aerodynamic diameter (MMAD) from about of from about 0.4 μmto about 5 μm.

In certain embodiments, the present disclosure provides a method oftreating, managing or lessening the severity of cystic fibrosis (CF)symptoms and infections in a subject, the method comprisingadministering to the subject a bismuth-thiol (BT) composition thatcomprises at least one BT compound, wherein the composition is asuspension of microparticles having a volumetric mean diameter (VMD)from about 0.4 μm to about 5 μm and/or a mass median aerodynamicdiameter (MMAD) from about 0.4 μm to about 5 μm.

In certain embodiments, the present disclosure provides a method oftreating, managing or lessening the severity of symptoms and infectionsassociated with one or more pulmonary diseases or infections in asubject, the method comprising administering to the subject abismuth-thiol (BT) composition that comprises at least one BT compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a representative inhalation exposure schematic.

FIG. 2 shows an aerosol particle size distribution of a 2.5 mg/mLsolution of BisEDT.

FIG. 3 shows an aerosol particle size distribution of a 25 mg/mLsolution of BisEDT.

FIG. 4 shows an aerosol particle size distribution of a 50 mg/mLsolution of BisEDT.

FIG. 5 shows an aerosol particle size distribution of a 75 mg/mLsolution of BisEDT.

FIG. 6 shows an aerosol particle size distribution of a 100 mg/mLsolution of BisEDT.

FIG. 7 shows an aerosol particle size distribution of a 100 mg/mL BisEDTin 300 mOsmolality phosphate buffer solution.

FIG. 8 shows an aerosol particle size distribution of a 50 mg/mL BisEDTin 300 mOsmolality phosphate buffer solution.

FIG. 9 shows an aerosol particle size distribution of a 10 mg/mL BisEDTin 300 mOsmolality phosphate buffer solution.

FIG. 10 shows an aerosol particle size distribution of a 2.5 mg/mLBisEDT in 300 mOsmolality phosphate buffer solution.

FIG. 11 shows that inhaled drug delivery of an antibiotic increases lungexposure (B) while reducing systemic exposure (A) of the correspondingside effects.

FIG. 12 shows the results of MIC testing of BisEDT against a variety ofclinically relevant CF isolates.

FIG. 13 is a diagram showing the evaluation of cytotoxicity through bothLDH release (from the culture medium side) and trans-epithelialelectrical resistance (TEER) from the apical/air-exposed side.

FIG. 14 shows the activity of BT compounds against biofilms grown fromMR14, which is a multidrug-resistant CF-isolate of Pseudomonasaeruginosa.

FIG. 15 shows the activity of BT compounds against biofilms grown fromAG14, which is an aminoglycoside-resistant CF-isolate of Pseudomonasaeruginosa.

FIG. 16 shows the activity of BT compounds against biofilms grown fromAU197, which is a CF-isolate of Burkholderia cenocepacia.

FIG. 17 shows the activity of BT compounds against biofilms grown fromAMT0130-8, which represents a CF-isolate of the clinically relevantMycobacterium abscessus complex (MAB SC), which frequently complicatesthe treatment of CF pulmonary infections

FIG. 18 shows the activity of BT compounds against biofilms grown fromAMT0089-5, which is a macrolide-resistant, amikacin-resistant MABSC.

FIG. 19 shows the activity of BT compounds against biofilms grown fromATCC-19977, which is M. abscessus (macrolide-resistant; inducible).

FIG. 20 shows the activity of BT compounds against biofilms grown fromMABSC CF isolate.

FIG. 21 shows the activity of BT compounds against biofilms grown fromAchromobacter spp.

FIG. 22 shows the activity of BT compounds against biofilms grown fromStenotrophomonas maltophilia.

FIG. 23 shows the activity of BT compounds against biofilms grown fromE. coli.

FIG. 24 shows an overview of MUCILAIR™ which is a fully differentiatedmodel of the human airway epithelia.

FIG. 25 shows the percentage of cytotoxicity (LDH measurement) of BisEDTin solution at 1, 8, 24 and 48 hours exposure. Bars representing 1, 8,24 and 48 hours are shown from left to right for each concentration.

FIG. 26 shows the effect on tissue integrity of BisEDT in solution at 1,8, 24 and 48 hours exposure. Bars representing 1, 8, 24 and 48 hours areshown from left to right for each concentration.

FIG. 27 shows the percentage of cytotoxicity (LDH measurement) of solidBisEDT at 1, 8, 24 and 48 hours exposure. Bars representing 1, 8, 24 and48 hours are shown from left to right for each concentration.

FIG. 28 shows the effect on tissue integrity of solid BisEDT at 1, 8, 24and 48 hours exposure. Bars representing 1, 8, 24 and 48 hours are shownfrom left to right for each concentration.

FIG. 29 shows the percentage of cytotoxicity (LDH measurement) of solidBisEDT at 1, 8, 24 and 48 hours exposure. Bars representing 1, 8, 24 and48 hours are shown from left to right for each concentration.

FIG. 30 shows the effect on tissue integrity of solid BisEDT at 1, 8, 24and 48 hours exposure. Bars representing 1, 8, 24 and 48 hours are shownfrom left to right for each concentration.

FIG. 31 shows the effect of sputum on the bacterial killing activity oftobramycin.

FIG. 32 shows that the bactericidal activity of BisEDT appears to bepartially inhibited by CF patient sputum.

FIG. 33 shows that the bactericidal activity of BisBDT appears to bepartially inhibited by CF patient sputum.

FIG. 34 shows a graph of lung tissue BisEDT concentration vs. time aftera single 100 μg/kg lung deposited dose in rats.

FIG. 35 shows whole blood BisEDT concentration vs. time (100 μg/kg IV or100 μg/kg inhalation or 250 μg/kg oral dose).

FIG. 36 shows rat blood BisEDT vs time after single inhalation dose(μg/kg lung deposited).

FIG. 37 shows rat lung BisEDT concentration (ng/g) at sacrifice (24 or30 hours after single inhaled dose).

FIG. 38 shows Particle Size Distribution for vehicle.

FIG. 39 shows Particle Size Distribution for Tobramycin.

FIG. 40 shows Particle Size Distribution for BisEDT.

FIG. 41 shows an example schematic diagram of the dog exposure system.

FIG. 42 shows rat efficacy figures showing cumulative (total)administered dose (lung deposited) at days 3 and 5.

FIG. 43 shows a representative checkerboard assay where each compound istested alone (Column 12 and Row H) and in combination at varying ratiosof drug concentration.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art ofthe present disclosure. The following references provide one of skillwith a general definition of many of the terms used in this disclosure:Singleton et al., Dictionary of Microbiology and Molecular Biology (2nded. 1994); The Cambridge Dictionary of Science and Technology (Walkered., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.),Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionaryof Biology (1991). As used herein, the following terms have the meaningsascribed to them below, unless specified otherwise.

As used herein, the verb “comprise” as is used in this description andin the claims and its conjugations are used in its non-limiting sense tomean that items following the word are included, but items notspecifically mentioned are not excluded. The present disclosure maysuitably “comprise”, “consist of”, or “consist essentially of”, thesteps, elements, and/or reagents described in the claims.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Throughout the present specification, the terms “about” and/or“approximately” may be used in conjunction with numerical values and/orranges. The term “about” is understood to mean those values near to arecited value. Furthermore, the phrases “less than about [a value]” or“greater than about [a value]” should be understood in view of thedefinition of the term “about” provided herein. The terms “about” and“approximately” may be used interchangeably.

An “alkyl” group or “alkane” is a straight chained or branchednon-aromatic hydrocarbon which is completely saturated. Typically, astraight chained or branched alkyl group has from 1 to about 20 carbonatoms, e.g. from 1 to about 10 unless otherwise defined. Examples ofstraight chained and branched alkyl groups include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl,pentyl and octyl. A C1-C6 straight chained or branched alkyl group isalso referred to as a “lower alkyl” group.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents, if nototherwise specified, can include, for example, a halogen, a hydroxyl, acarbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl),a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. It will be understood by those skilled in the artthat the moieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl can include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF3, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF3, —CN, and the like.

The term “Cx-y” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. Forexample, the term “Cx-yalkyl” refers to substituted or unsubstitutedsaturated hydrocarbon groups, including straight-chain alkyl andbranched-chain alkyl groups that contain from x to y carbons in thechain, including haloalkyl groups such as trifluoromethyl and2,2,2-tirfluoroethyl, etc. C0 alkyl indicates a hydrogen where the groupis in a terminal position, a bond if internal. The terms “C2-yalkenyl”and “C2-yalkynyl” refer to substituted or unsubstituted unsaturatedaliphatic groups analogous in length and possible substitution to thealkyls described above, but that contain at least one double or triplebond respectively.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and can be represented by the generalformula alkylS—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein each R³¹ independently represents a hydrogen or a hydrocarbylgroup, or two R³¹ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure. The term “aminoalkyl”, as used herein, refers to an alkylgroup substituted with an amino group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon. Insome embodiments, the ring is a 5- to 7-membered ring, e.g. a 6-memberedring. The term “aryl” also includes polycyclic ring systems having twoor more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is aromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene,naphthalene, phenanthrene, phenol, aniline, and the like.

The term “bismuth” refers to the 83^(rd) element of the periodic table,or atoms or ions thereof. Bismuth can occur in the metallic state or inthe ionized state, such as in the III or V oxidation state. Bismuth ionscan form complexes with anions, either to make bismuth salts, or to formcomplex anions which are then further complexed with one or moreadditional cation(s). Bismuth can also form covalent bonds to otheratoms, such as sulfur.

As disclosed herein, a “bismuth-thiol compound” or “BT compound” is acompound that has a bismuth atom covalently bound to one, two or threeother sulfur atoms present on one or more thiol compounds. The term“thiol” refers to a carbon-containing compound, or fragment thereof,containing an —SH group and can be represented by the general formulaR—SH. These thiol compounds include compounds with one, two, three ormore S atoms. Thiol compounds can have other functionality, such asalkyl, hydroxyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, amino, andother substituents. Thiol compounds having two or more S atoms canchelate the bismuth atom, such that two S atoms from the same moleculecovalently bond with the bismuth atom. Exemplary bismuth-thiol compoundsare shown below:

The terms “carbocycle”, and “carbocyclic”, as used herein, refers to asaturated or unsaturated ring in which each atom of the ring is carbon.The term carbocycle includes both aromatic carbocycles and non-aromaticcarbocycles. Non-aromatic carbocycles include both cycloalkane rings, inwhich all carbon atoms are saturated, and cycloalkene rings, whichcontain at least one double bond.

The term “carbocycle” includes 5-7 membered monocyclic and 8-12 memberedbicyclic rings. Each ring of a bicyclic carbocycle can be selected fromsaturated, unsaturated and aromatic rings. Carbocycle includes bicyclicmolecules in which one, two or three or more atoms are shared betweenthe two rings. The term “fused carbocycle” refers to a bicycliccarbocycle in which each of the rings shares two adjacent atoms with theother ring. Each ring of a fused carbocycle can be selected fromsaturated, unsaturated and aromatic rings. In an exemplary embodiment,an aromatic ring, e.g., phenyl, can be fused to a saturated orunsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Anycombination of saturated, unsaturated and aromatic bicyclic rings, asvalence permits, is included in the definition of carbocyclic. Exemplary“carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane,1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fusedcarbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene andbicyclo[4.1.0]hept-3-ene. “Carbocycles” can be substituted at any one ormore positions capable of bearing a hydrogen atom.

A “cycloalkyl” group is a cyclic hydrocarbon which is completelysaturated. “Cycloalkyl” includes monocyclic and bicyclic rings.Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbonatoms, more typically 3 to 8 carbon atoms unless otherwise defined. Thesecond ring of a bicyclic cycloalkyl can be selected from saturated,unsaturated and aromatic rings. Cycloalkyl includes bicyclic moleculesin which one, two or three or more atoms are shared between the tworings. The term “fused cycloalkyl” refers to a bicyclic cycloalkyl inwhich each of the rings shares two adjacent atoms with the other ring.The second ring of a fused bicyclic cycloalkyl can be selected fromsaturated, unsaturated and aromatic rings. A “cycloalkenyl” group is acyclic hydrocarbon containing one or more double bonds.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The term “heteroalkyl”, as used herein, refers to a saturated orunsaturated chain of carbon atoms and at least one heteroatom, whereinno two heteroatoms are adjacent.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, for example 5- to7-membered rings, e.g. 5- to 6-membered rings, whose ring structuresinclude at least one heteroatom, for example one to four heteroatoms,e.g. one or two heteroatoms. The terms “heteroaryl” and “hetaryl” alsoinclude polycyclic ring systems having two or more cyclic rings in whichtwo or more carbons are common to two adjoining rings wherein at leastone of the rings is heteroaromatic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heteroaryl groups include, for example, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine,pyridazine, and pyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, for example,3- to 10-membered rings, more e.g. 3- to 7-membered rings, whose ringstructures include at least one heteroatom, e.g. one to fourheteroatoms, e.g. one or two heteroatoms. The terms “heterocyclyl” and“heterocyclic” also include polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings wherein at least one of the rings is heterocyclic, e.g., the othercyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, forexample, piperidine, piperazine, pyrrolidine, morpholine, lactones,lactams, and the like.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but can optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer non-hydrogen atoms in thesubstituent, for example, six or fewer. A “lower alkyl”, for example,refers to an alkyl group that contains ten or fewer carbon atoms, e.g.six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl,alkynyl, or alkoxy substituents defined herein are respectively loweracyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or loweralkoxy, whether they appear alone or in combination with othersubstituents, such as in the recitations hydroxyalkyl and aralkyl (inwhich case, for example, the atoms within the aryl group are not countedwhen counting the carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, e.g. from 5 to 7.

The term “N-oxide” refers to a zwitterionic group containing a nitrogenatom in the +1 oxidaton state bound to an oxygen atom in the −1oxidation state. An non-limiting example of an N-oxide is pyridiumN-oxide shown below. As used herein, the term “N-oxide” encompassessubstituents of other groups having this functionality.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms such as nitrogen canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxy, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that substituents canthemselves be substituted, if appropriate. Unless specifically stated as“unsubstituted,” references to chemical moieties herein are understoodto include substituted variants. For example, reference to an “aryl”group or moiety implicitly includes both substituted and unsubstitutedvariants.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group. A “thiol compound” as discussed abovecan include a thioalkyl as a substituent on the compound structure. Athiol compound can have, for example, one, two, three or more thioalkylgroups.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The term “subject” to which administration is contemplated includes, butis not limited to, humans (i.e., a male or female of any age group,e.g., a pediatric subject (e.g., infant, child, adolescent) or adultsubject (e.g., young adult, middle-aged adult or senior adult)) and/orother primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals,including commercially relevant mammals such as cattle, pigs, horses,sheep, goats, cats, and/or dogs; and/or birds, including commerciallyrelevant birds such as chickens, ducks, geese, quail, and/or turkeys.Preferred subjects are humans.

As used herein, the phrase “conjoint administration” refers to any formof administration of two or more different therapeutic compounds suchthat the second compound is administered while the previouslyadministered therapeutic compound is still effective in the body (e.g.,the two compounds are simultaneously effective in the patient, which mayinclude synergistic effects of the two compounds). For example, thedifferent therapeutic compounds can be administered either in the sameformulation or in a separate formulation, either concomitantly orsequentially. In certain embodiments, the different therapeuticcompounds can be administered within one hour, 12 hours, 24 hours, 36hours, 48 hours, 72 hours, or a week.

“Coadministration” refers to the administration of the two agents in anymanner in which the pharmacological effects of both agents are manifestin the patient at the same time. Thus, concomitant administration doesnot require that a single pharmaceutical composition, the same dosageform, or even the same route of administration be used foradministration of both agents or that the two agents be administered atprecisely the same time. However, in some situations, coadministrationwill be accomplished most conveniently by the same dosage form and thesame route of administration, at substantially the same time.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

The term “treating” means one or more of relieving, alleviating,delaying, reducing, improving, or managing at least one symptom of acondition in a subj ect. The term “treating” may also mean one or moreof arresting, delaying the onset (i.e., the period prior to clinicalmanifestation of the condition) or reducing the risk of developing orworsening a condition.

The term “managing” includes therapeutic treatments as defined above.Managing includes achieving a steady state level of infection asdetermined by known methods in the art. The steady state can includeevaluation of one or more of the severity of the infection(s), the sizeand location of the infection(s), the number of different microbialpathogens present in the infection(s), the level of antibiotic tolerantor resistant microbial pathogens, the degree of response to treatment,such as with a BT composition disclosed herein, the degree of biofilmformation and reduction, and the side effects experienced by thesubject. During management of an infection, the infection may fluctuatefrom increasing to lessening in severity, in the amount or extent ofinfection, amount of side effects experienced by the subject, or othersubject outcome indicia. Over a period of time, such as days, month, oryears, the degree of management of the infection can be determined byevaluation of the above factors to assess whether the clinical course ofinfection has improved, is bacteriostatic, or has worsened. In someembodiments, managing an infection include successful treatment ofmicrobial pathogen(s) that are otherwise drug tolerant or drugresistant.

The term “lessen the severity” of infection(s) refers to an improvementin the clinical course of the infection on any measurable basis. Suchbasis can include measurable indices such as reducing the extent ofinfection (s), whether the infection(s) are considered acute, the numberand identity of microbial pathogens causing the infection(s), the extentof microbial (e.g. bacterial or fungal) biofilms, and side effectsexperienced by the subject. In some embodiments, lessening the severityof an infection is determined by measurements such as reduction insputum infection counts (e.g. a reduction in CFU in the sputum). In someembodiments, lessening the severity involves halting a steady decline inoutcome to achieve stabilized infection(s), resulting in the subjectentering successful management of the infection(s). In otherembodiments, lessening the severity can result in substantial tocomplete treatment of the infection(s). In some embodiments, lesseningthe severity refers to a lessening of exacerbations associated with thedisease or infection (for example by a 1%-99% decrease inexacerbations). In some embodiments, lessening the severity can refer toan increase in lung function (for example by a 1%-99% increase in lungfunction).

As used herein, the term “exacerbation” refers to an increase in theseverity of symptoms during a course of a disease which is mostlyassociated with a worsening of quality of life. Exacerbations are quitefrequent in patients with chronic lung diseases such as CF. Bydefinition, exacerbations are simply a worsening and/or increase insymptoms.

In some embodiments, lessening the severity of infections and/orsymptoms can relate to patient-reported outcomes (“PROSs”). A PROinstrument is defined as any measure of a subject's health status thatis elicited from the patient and determines how the patient “feels orfunctions with respect to his or her health condition.” PROs areparticularly useful in reporting outcomes in CF and whether the severityof symptoms has been reduced or lessened. Such symptoms can beobservable events, behaviors, or feelings (e.g., ability to walkquickly, lack of appetite, expressions of anger), or unobservableoutcomes that are known only to the patient (e.g., perceptions of pain,feelings of depression).

An “effective amount”, as used herein, refers to an amount that issufficient to achieve a desired biological effect. A “therapeuticallyeffective amount”, as used herein refers to an amount that is sufficientto achieve a desired therapeutic effect. For example, a therapeuticallyeffective amount can refer to an amount that is sufficient to improve atleast one sign or symptom of infection (e.g. respiratory infection).

A “response” to a method of treatment can include a decrease in oramelioration of negative symptoms, a decrease in the progression of aninfection or symptoms thereof, an increase in beneficial symptoms orclinical outcomes, a lessening of side effects, stabilization of theinfection, and partial or complete remedy of infection, among others.

“Antibiotic susceptibility or sensitivity” refers to whether a bacteriawill be successfully treated by a given antibiotic. Similarly,“Antifungal susceptibility or sensitivity” refers to whether a fungiwill be successfully treated by a given antibiotic. Testing forsusceptibility can be performed by methods known in the art such as theKirby-Bauer method, the Stokes method and Agar Broth dilution methods.The effectiveness of an antibiotic in killing the bacteria or preventingbacteria from multiplying can be observed as areas of reduced or stableamount, respectively, of bacterial growth on a medium such as a wafer,agar, or broth culture.

“Antimicrobial tolerance” refers to the ability of a microbe, such asbacteria or fungi, to naturally resist being killed by antibiotics. Itis not caused by mutant microbes but rather by microbial cells thatexist in a transient, dormant, non-dividing state. Antibiotic or drugtolerance is caused by a small subpopulation of microbial cells termedpersisters. Persisters are not mutants, but rather are dormant cellsthat can survive the antimicrobial treatments that kill the majority oftheir genetically identical siblings. Persister cells have entered anon- or extremely slow-growing physiological state which makes theminsensitive (refractory or tolerant) to the action of antimicrobialdrugs. Similarly, “antibiotic tolerance” refers to the ability of abacteria to naturally resist being killed by antibiotics and “antifungaltolerance” refers to the ability of a fungi to naturally resist beingkilled by antibiotics.

“Antimicrobial resistance” refers to the ability of a microbe to resistthe effects of medication that once could successfully treat themicrobe. Microbes resistant to multiple antimicrobials are calledmultidrug resistant (MDR). Resistance arises through one of threemechanisms: natural resistance in certain types of bacteria, geneticmutation, or by one species acquiring resistance from another. Mutationscan lead to drug inactivation, alteration of the drug's binding site,alteration of metabolic pathways and decreasing drug permeability.

As used herein, the terms “antibacterial activity”, “antifungalactivity” and “antimicrobial activity”, with reference to a BTcomposition of the present disclosure, refers to the ability to killand/or inhibit the growth or reproduction of a particular microorganism.In certain embodiments, antibacterial or antimicrobial activity isassessed by culturing bacteria, e.g., Gram-positive bacteria (e.g., S.aureus), Gram-negative bacteria (e.g., A. baumannii, E. coli, and/or P.aeruginosa) or bacteria not classified as either Gram-positive orGram-negative, or fungi according to standard techniques (e.g., inliquid culture or on agar plates), contacting the culture with a BTcomposition of the present disclosure and monitoring cell growth aftersaid contacting. For example, in a liquid culture, bacteria may be grownto an optical density (“OD”) representative of a mid-point inexponential growth of the culture; the culture is exposed to one or moreconcentrations of one or more BT compounds of the present disclosure, orvariants thereof, and the OD is monitored relative to a control culture.Decreased OD relative to a control culture is representative ofantibacterial activity (e.g., exhibits lytic killing activity).Similarly, bacterial colonies can be allowed to form on an agar plate,the plate exposed to a BT composition of the present disclosure, orvariants thereof, and subsequent growth of the colonies evaluatedrelated to control plates. Decreased size of colonies, or decreasedtotal numbers of colonies, indicate antibacterial activity.

“Biofilm” refers any syntrophic consortium of microorganisms in whichcells stick to each other and often also to a surface. These adherentcells become embedded within a slimy extracellular matrix that iscomposed of extracellular polymeric substances (EPS). Upon formation ofbiofilms, microbial resistance to antibiotics is up to 1000 timesgreater compared to that of planktonic bacteria. Bacterial aggregatesare clusters of laterally aligned cells can initiate biofilmdevelopment, which has a more complex and denser 3-D structure. In someembodiments, the biofilm may comprise one or more species of bacteria(e.g., Pseudomonas aeruginosa and Staphylococcus aureus) and/or one ormore different phyla (e.g., bacteria, virus and fungi).

As used herein, discussion of bacterial or fungal pathogens alsoencompass any microbe (bacteria and/or fungi) that contributes to thepathological state in the lungs. This includes both recognized andunrecognized microbes, and may also include bacteria or fungi that arenot pathogens, but that simply facilitate the activity and presence ofpathogens and their biofilms. As an example, embodiments directed to theinhibition of cell viability or cell growth of planktonic cells of thebacterial or fungal pathogen also extend to the inhibition of cellviability or cell growth of planktonic cells of the bacterial and/orfungal microbes that simply facilitate the activity and presence ofpathogens and their biofilms.

“Airway surface” and “pulmonary surface,” as used herein, includepulmonary airway surfaces such as the bronchi and bronchioles, alveolarsurfaces, and nasal and sinus surfaces.

“Saline” as used herein refers to a solution comprised of, consistingof, or consisting essentially of sodium chloride in water. Saline can behypertonic, isotonic, or hypotonic. In some embodiments, saline cancomprise sodium chloride in an amount of from about 0.1% to about 40% byweight, or any range therein, such as, but not limited to, about 0.1% toabout 10%, about 0.5% to about 15%, about 1% to about 20%, about 5% toabout 25%, about 10% to about 40%, or about 15% to about 35% by weight(in mg/100 mL). In certain embodiments, sodium chloride is included in asolution in an amount of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 3 4%, 35%, 36%, 37%, 38%, 39%, 40% by weight(in mg/100 mL), or any range therein.

“Hypertonic saline” as used herein refers to a solution comprised of,consisting of, or consisting essentially of greater than 0.9 wt % sodiumchloride in water. In general, the sodium chloride is included in thesolution in an amount of from about 0.9% to about 40% by weight, or anyrange therein, such as, but not limited to, about 1% to about 15%, about5% to about 20%, about 5% to about 25%, about 10% to about 40%, or about15% to about 35% by weight. In certain embodiments, sodium chloride isincluded in the solution in an amount of about 0.9%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40% by weight, or any range therein.

“Hypotonic saline” as used herein refers to a solution comprised of,consisting of, or consisting essentially of less than 0.9 wt % sodiumchloride in water. In some embodiments, sodium chloride is included inthe solution in an amount of about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,0.3%, 0.2%, 0.1% by weight, or any range therein.

“Isotonic saline” as used herein refers to a solution comprised of,consisting of, or consisting essentially of 0.9 wt % sodium chloride inwater.

According to some embodiments, saline (e.g., hypertonic saline) caninclude an excipient. An excipient can be a pharmaceutically acceptableexcipient. “Pharmaceutically acceptable” as used herein means that thecompound or composition is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the severity of the disease and necessity of thetreatment. Exemplary excipients include, but are not limited to, abuffer and/or a buffering agent (e.g., an anion, a cation, an organiccompound, a salt, etc.). Exemplary buffers include, but are not limitedto, carbonic acid/carbonate/bicarbonate-based buffers, disodium hydrogenphthalate/sodium dihydrogen orthophosphate-based buffers,tris(hydroxymethyl)aminomethane/hydrochloric acid-based buffers,barbitone sodium/hydrochloric acid-based buffers, and any combinationthereof. Exemplary buffering agents include, but are not limited to,carbonic acid, carbonate, bicarbonate, disodium hydrogen phthalate,sodium dihydrogen orthophosphate, tris(hydroxymethyl)aminomethane,hydrochloric acid, barbitone sodium, dissolved CO₂ (e.g., CO₂ formulatedat a pH of greater than 6.6), and any combination thereof. In certainembodiments, saline comprises a bicarbonate buffer excipient, such as abicarbonate anion (HCO₃). In some embodiments, hypertonic saline caninclude sodium bicarbonate, sodium carbonate, carbonic acid, and/ordissolved CO2 formulated at a pH of greater than 6.5. Additionalingredients can be included as desired depending upon the particularcondition being treated, as discussed further below.

As used herein, the term “volumetric median diameter” or “VMD” of anaerosol is the particle size diameter identified such that half of themass of the aerosol particles is contained in particles with largerdiameter than the VMD, and half of the mass of the aerosol particles iscontained in particles with smaller diameter than the VMD. VMD istypically measured by laser diffraction.

“Mass median aerodynamic diameter” or “MMAD” is a measure of theaerodynamic size of a dispersed aerosol particle. The aerodynamicdiameter is used to describe an aerosolized particle in terms of itssettling behavior, and is the diameter of a unit density sphere havingthe same settling velocity, generally in air, as the particle inquestion. The aerodynamic diameter encompasses particle shape, densityand physical size of a particle. As used herein, MMAD refers to themidpoint or median of the aerodynamic particle size distribution of anaerosolized particle determined by cascade impaction and/or laser timeof flight and/or cascade impactor.

“Mass median diameter” or “MMD” is a measure of mean particle size. Anynumber of commonly employed techniques can be used for measuring meanparticle size.

As used herein, “D90” refers to the 90% value of particle diameter(either the microparticle or aerosolized particle). For example if D90=1μm, 90% of the particles are smaller than 1 μm. Similarly, “D80” refersto the 80% value of particle diameter (either the microparticle oraerosolized particle), “D70” refers to the 70% value of particlediameter (either the microparticle or aerosolized particle), “D60”refers to the 60% value of particle diameter (either the microparticleor aerosolized particle), “D50” refers to the 50% value of particlediameter (either the microparticle or aerosolized particle), “D40”refers to the 40% value of particle diameter (either the microparticleor aerosolized particle), “D30” refers to the 30% value of particlediameter (either the microparticle or aerosolized particle), “D20”refers to the 20% value of particle diameter (either the microparticleor aerosolized particle), “D10” refers to the 10% value of particlediameter (either the microparticle or aerosolized particle).

As used herein, “monodisperse” refers to a collection of particles (bulkor aerosol dispersion) comprising particles of a substantially uniformMMD and/or MMAD and/or VMD.

As used herein, the term “deposition efficiency” refers to thepercentage of the delivered dose that is deposited into the area ofinterest. Thus, the deposition efficiency of a method and/or system fordelivering an aerosolized medicament into the lungs is the amount bymass of the aerosol deposited into the lungs divided by the total amountof the aerosol delivered by the system to the nares.

As used herein, “substantially” or “substantial” refers to the completeor nearly complete extent or degree of an action, characteristic,property, state, structure, item, or result. For example, an object thatis “substantially” enclosed would mean that the object is eithercompletely enclosed or nearly completely enclosed. The exact allowabledegree of deviation from absolute completeness may in some cases dependon the specific context. However, generally speaking, the nearness ofcompletion will be so as to have the same overall result as if absoluteand total completion were obtained. The use of “substantially” isequally applicable when used in a negative connotation to refer to thecomplete or near complete lack of action, characteristic, property,state, structure, item, or result. For example, a composition that is“substantially free of” other active agents would either completely lackother active agents, or so nearly completely lack other active agentsthat the effect would be the same as if it completely lacked otheractive agents. In other words, a composition that is “substantially freeof” an ingredient or element or another active agent may still containsuch an item as long as there is no measurable effect thereof.

Methods of Use

Cystic fibrosis (CF), an autosomal recessive disorder, is caused byfunctional deficiency of the cAMP-activated plasma membrane chloridechannel, cystic fibrosis transmembrane conductance regulator (CFTR),which results in pulmonary and other complications.

In cystic fibrosis patients, the absence or dysfunction of CFTR leads toexocrine gland dysfunction and a multisystem disease, characterized bypancreatic insufficiency and malabsorption, as well as abnormalmucociliary clearance in the lung, mucostasis, chronic lung infectionand inflammation, decreased lung function and ultimately respiratoryfailure.

The loss of a functional CFTR channel at the plasma membrane disruptsionic homeostasis and airway surface hydration leading to reduced lungfunction. Reduced periciliary liquid volume and increased mucusviscosity impede mucociliary clearance resulting in chronic infectionand inflammation.

In healthy individuals, clearance of lung bacteria relies on theconcerted action of two anatomic features: (i) the ciliated apicalsurface of the airway epithelium and (ii) a mucus layer that lines theairway lumen. The airway cilia beat synchronously, creating a steadycurrent that continually moves the mucus layer upward toward thenasopharynx. The mucus layer is biphasic, consisting of an upper,viscous layer that serves to trap particulates and microorganisms and alower, more fluid layer in which the cilia beat. When functioningnormally, this clearance system traps foreign bodies in the mucus andsubsequently carries them to the nasopharynx, where they areexpectorated and swallowed.

However, abnormal secretory characteristics of the CF airway cells dueto the ion imbalance caused by the mutant CFTR protein alter theviscosity of the airway fluid, such that the normally serous“periciliary” layer becomes thicker, inhibiting escalator action thatclears foreign bodies. Bacteria are trapped in the mucous and result inan ongoing infection in the lungs.

CF patients routinely produce sputum from the lungs through coughing,aided by other physical therapies designed to free mucous from thelungs. Many of the organisms that are isolated from CF sputum arepathogens that often benignly colonize the upper respiratory tract(e.g., non-typeable H. influenzae) or the nose (e.g., S. aureus) or arecommon environmental organisms that behave as pathogens only undercertain opportunistic situations (e.g., P. aeruginosa). Differentbacteria and the level of infection in the lungs can be determinative ofa CF patient's symptoms and outcome. For example, the presence of S.aureus and the absence of P. aeruginosa predicts long term survival inCF patients after the age of 18 years. In addition, the potential forincreasing P. aeruginosa colonization as a consequence of suppression ofS. aureus infection may be relevant for some patients.

Of all the bacteria that can colonize in the lungs of CF patients,chronic P. aeruginosa airway infection and the accompanying inflammatoryresponse are the major clinical problems for CF patients today. Whileantibiotic chemotherapy and chemoprophylaxis have reduced the morbidityand early mortality of CF patients from this infection, the intrinsicability of P. aeruginosa to develop resistance to many commonly usedantibiotics probably contributes to the inability to eradicate P.aeruginosa from CF patients' lung and ultimately allows this microbe tobe highly problematic for these patients.

CF patients can acquire P. aeruginosa in their respiratory tracts at anytime, with most studies indicating that 70 to 80% CF patients areinfected by their teen years. P. aeruginosa infection probably initiallyoccurs within the first 3 years of life. After the onset of chronicinfection, patients experience episodic exacerbations that can benefitfrom antibiotic chemotherapy. Infection may result from social contactsor may be hospital acquired, but the diversity of P. aeruginosa clonesisolated from CF patients suggests that most clinical isolates originatein the environment. CF patients chronically infected by P. aeruginosashow a steeper lung function decline (expressed as forced expired volumein 1 second (FEV1) decline over time), a higher number of pulmonaryexacerbations, more hospital admissions and higher mortality than P.aeruginosa-free patients. The effects of P. aeruginosa are more severeif chronic infection develops early.

P. aeruginosa infections can change over time to develop a mucoidphenotype, which can initiate the chronic-infection stage of cysticfibrosis. The mucoid phenotype results from bacterial production of apolysaccharide known as both alginate and mucoid exopolysaccharide (MEP)and plays an important role in bacterial evasion of the host immuneresponse. The MEP/alginate itself is able to promote bacterial survivalin the face of host immune effectors. Alginate overproduction by P.aeruginosa correlates with the onset of significant deterioration inlung function. In addition, P. aeruginosa can grow as a biofilm, whichincreases bacterial resistance to phagocytic action and antibioticefficacy.

Bacterial biofilms are a matrix of cells that adhere to each other andoften a surface, such as lung mucosa. The bacterial cells becomeembedded within an extracellular matrix formed from extracellularpolymeric substances, such as polysaccharides, proteins, lipids and DNA.Biofilm bacterial cells are physiologically different than planktoniccells in which a large number of genes are differentially regulated.Biofilms can also be more resistant to antibiotics given the shelterprovided by the matrix. Biofilms of P. aeruginosa and other bacteriathat are present in the lungs of CF patients increase the difficulty ofsuccessful infection management and reduction. Combinations ofCF-relevant bacteria forming multispecies biofilms containing P.aeruginosa have demonstrated greater resistance, virulence andpathogenicity than comparable single-species biofilms. The presence ofsuch complex biofilms in the lungs of CF patients is considered to belargely responsible for the chronic, persistent nature of thesepulmonary infections, which are not only responsible for chronic,ongoing and progressive morbidity, but are also ultimately responsiblefor mortality in this population.

In addition to P. aeruginosa, other pathogens commonly found in CFpatients' lungs include, but are not limited to, Haemophilus influenzae,Staphylococcus aureus, Staphylococcus warneri, Staphylococcuslugdunensis, Staphylococcus epidermidis, Streptococcus milleri/anginous,Streptococcus pyogenes, non-tuberculosis mycobacterium, Mycobacteriumtuberculosis, Burkholderia spp., Achromobacter xylosoxidans, Pandoraeasputorum, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans,Haemophilus pittmaniae, Serratia marcescens, Candida albicans, drugresistant Candida albicans, Candida glabrata, Candida krusei, Candidaguilliermondii, Candida auris, Candida tropicalis, Aspergillus niger,Aspergillus terreus, Aspergillus fumigatus, Aspergillus flavus,Morganella morganii, Inquilinus limosus, Ralstonia mannitolilytica,Pandoraea apista, Pandoraea pnomenusa, Pandoraea sputorum, Bdellovibriobacteriovorus, Bordetella bronchiseptica, Vampirovibrio chlorellavorus,Actinobacter baumanni, Cupriadidus metallidurans, Cupriavidus pauculus,Cupriavidus respiraculi, Delftia acidivordans, Exophilia dermatitidis,Herbaspirillum frisingense, Herbaspirillum seropedicae, Klebsiellapneumoniae, Pandoraea norimbergensis, Pandoraea pulmonicola, Pseudomonasmendocina, Pseudomonas pseudoalcaligenes, Pseudomonas putida,Pseudomonas stutzeri, Ralstonia insidiosa, Ralstonia pickettii,Neisseria gonorrhoeae, NDM-1 positive E. coli, Enterobacter cloaca,Vancomycin-resistant E. faecium, Vancomycin-resistant E. faecalis, E.faecium, E. faecalis, Clindamycin-resistant S. agalactiae, S.agalactiae, Bacteroides fragilis, Clostridium difficile, Streptococcuspneumonia, Moraxella catarrhalis, Haemophilus haemolyticus, Haemophilusparainfluenzae, Chlamydophilia pneumoniae, Mycoplasma pneumoniae,Atopobium spp., Sphingomonas spp., Saccharibacteria spp., Leptotrichiaspp., Capnocytophaga, Oribacterium spp., Aquabacterium spp.,Lachnoanaerobaculum spp., Campylobacter spp., Acinetobacter spp.,Agrobacterium spp., Bordetella spp., Brevundimonas spp.,Chryseobacterium spp., Delftia spp., Enterobacter spp., Klebsiella spp.,Pandoraea spp., Pseudomonas spp., Ralstonia spp., and Prevotella spp.

Exemplary non-tuberculosis mycobacterium include, but are not limitedto, Mycobacterium abscessus, Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium fortuitum, Mycobacterium gordonae,Mycobacterium kansasii, Mycobacterium avium complex (MAC), Mycobacteriumabscessus complex (MABSC) Mycobacterium marinum, Mycobacterium terraeand Mycobacterium cheloni.

Exemplary species of Burkholderia include, but are not limited to,Burkholderia cepacia, Burkholderia multivorans, Burkholderiacenocepacia, Burkholderia stabilis, Burkholderia vietnamiensis,Burkholderia dolosa, Burkholderia ambifaria, Burkholderia anthina,Burkholderia pyrrocinia, Burkholderia gladioli, Burkholderia ubonensis,Burkholderia arboris, Burkholderia latens, Burkholderia lata,Burkholderia metallica, Burkholderia seminalis, Burkholderiacontaminans, and Burkholderia diffusa.

In some embodiments, the bacterial pathogen is selected from Pseudomonasaeruginosa, multi drug-resistant Pseudomonas aeruginosa, Staphylococcusaureus, multi drug-resistant Staphylococcus aureus, methicillinresistant Staphylococcus aureus, Mycobacterium abscessus, Mycobacteriumavium, Burkholderia cepacia, Burkholderia multivorans, Burkholderiacenocepacia, Burkholderia dolosa, Achromobacter xylosoxidans,Stenotrophomonas maltophilia Staphylococcus epidermidis, andBurkholderia vietnamiensis. In certain embodiments, the bacterialpathogen is selected from Haemophilus influenzae, Pseudomonasaeruginosa, and Staphylococcus aureus. In certain embodiments, thebacterial pathogen is selected from biofilms of Pseudomonas aeruginosa,Burkholderia cenocepacia, Burkholderia cepacia complex, Mycobacteriumabscessus, Mycobacterium avium, Achromobacter spp., Staphylococcusepidermidis, Stenotrophomonas maltophilia, and Staphylococcus aureus.

In some embodiments, the bacterial pathogen exhibits resistance to oneor more antibiotics. Methicillin-resistant S. aureus (MRSA) is anexample of a singly resistant strain that is difficult to treat in CFpatients and the population at large, while even more challengingmulti-drug resistant (MDR) strains can occur in bacteria such as P.aeruginosa and S. aureus. For example, a bacterial pathogen can becomeresistant to known standards of antibiotic care, including, but notlimited to, amikacin, aztreonam, methicillin, vancomycin, nafcillin,gentamicin, ampicillin, chloramphenicol, doxycycline and tobramycin. Insome embodiments, the resistant antibiotic is amikacin, aztreonam, ortobramycin.

Long-term, repeated treatment with antibiotics to treat CF-associatedinfections typically results in development of antibiotic-resistance,characterized by the presence of microbial biofilms. Recent research hasrepeatedly demonstrated a correlation between multi-drug resistant (MDR)bacteria, and stronger, more prolific biofilm-forming capabilities.Biofilm involvement in the lung is considered highly immunogenic,accelerating structural lung damage. Further, bacteria within biofilmsare protected from antibiotics, which increases the minimal inhibitoryconcentration of such antibiotics. Biofilms tend to reduce theantimicrobial activity of aminoglycosides and beta-lactam antibiotics byboth changing the pH of the respiratory mucosa and through theproduction of beta-lactamase enzymes. The involvement of biofilm-formingbacteria in CF is correlated with decreased lung function and reducedQuality of Life, decreased response to antibiotic therapy, increasedexacerbations, and, over time, reduced survival.

In some embodiments, the BT composition is administered by inhalation,either orally or nasally, using an aerosol device, such as a nebulizer.A nebulizer can administer the BT composition topically to the lungtissue, which can include the lung mucosa, the alveoli (e.g. deep lungalveoli), the bronchi and/or the bronchioles. Thus, in some embodiments,the present disclosure provides for administration of the BT compositionto the deep lung region of the lung (e.g. the deep lung alveoli). Localtopical administration of the BT composition provides several keyadvantages over systemic antibiotic therapies. The term “systemic”refers to administration of a medication into the circulatory system ofthe subject such that the majority of the entire body can be exposed.Systemic administration of a medication can occur enterally (absorptionthrough the gastrointestinal tract, e.g. oral administration) orparenterally (absorption through injection or infusion, e.g.intravenously).

Systemic anti-infective products have several disadvantages relating tothe treatment of localized infections, including: (a) difficulty inachieving a therapeutically effective concentration at the site of localinfection, particularly in the case of infections associated withtopical locations, such as pulmonary airways, (b) frequent unintendedtoxic effects on organ systems exposed through systemic circulation, and(c) increased generation of antibiotic-resistant bacteria as a result ofthe widespread exposure of the body's normal flora to the anti-infectiveagent, (d) resulting in greater likelihood of transmission of suchantibiotic-resistant bacteria to others as a result of long termsustained/repeated exposure of the entire body's complement of normalflora and (e) reduced preservation of the beneficial influence of thehealthy normal flora throughout the body due to extensive systemicexposure.

In FIG. 11 , plasma concentration is depicted for an orally andpulmonary dosed drug. Oral dosing (A) can result in high plasmaconcentrations that may lead to toxicity and varied inter-patientexposure (variable absorption and variable first-pass, hepaticclearance) or drug-drug interactions. High plasma exposure is necessaryto achieve therapeutic exposure in the lungs. In contrast, inhaleddosing for topical lung indications requires a lower total dose toachieve an efficacious MIC, which results in significantly less systemicexposure. Inhaled dosing (B) achieves higher local concentrations in thelung that significantly exceed the MIC of the drug over a long period oftime. Due to the delivery of high concentrations of drug directly to thelung, the achieved pulmonary concentrations following inhalation maygreatly exceed those achieved by oral dosing. From previous experiencewith inhaled antibiotics, the lung concentration of drug upon inhalationis >100× greater than upon systemic/oral administration of the samedose.

In some embodiments, one or more of the following symptoms (e.g. cysticfibrosis-related symptoms) is lessened in severity in the subject:cough, wheezing, breathlessness, bronchiectasis, nasal polyps,hemoptysis, respiratory failure, and pulmonary exacerbation. Additionalnon-cystic fibrosis related infections may also be lessened in severity.FIG. 11 demonstrates the differences in side effect severity andefficacy of an inhaled antibiotic vs. one delivered systemically. Thus,inhaled formulations of the BT compositions disclosed herein provide amore targeted and effective antibiotic treatment than a correspondingformulation administered systemically.

BT compounds are known broad-spectrum antimicrobial (and anti-biofilm)small molecule drug product for the treatment of chronic, ultimatelylife-threatening pulmonary infections secondary to CF. Its efficacyextends to Gram-positive, antibiotic-resistant pathogens includingmethicillin-resistant Staphylococcus aureus (MRSA, includingcommunity-associated [CA]-MRSA), methicillin-resistant Staphylococcusepidermidis (MRSE), and vancomycin-resistant Enterococcus (VRE). BTcompounds are also potent against Multi-drug-resistant (MDR)Gram-negative pathogens including Pseudomonas aeruginosa, Escherichiacoli, Klebsiella pneumoniae (including, in all of the afore-mentionedbacteria, carbapenem-resistant strains), and Acinetobacter baumannii.

BT compounds have the dual ability to overcome a) a very diversifiedspectrum of antibiotic resistance profiles (due toevolution/diversification driven by persistence, time and isolation inmany different anatomical regions throughout the pulmonary airways), andb) antibiotic-resistant and MDR biofilms.

Disclosed herein are methods of treating, managing or lessening theseverity of cystic fibrosis (CF) symptoms and infections in a subject,the method comprising administering to the subject a bismuth-thiol (BT)composition that comprises at least one BT compound. Also disclosedherein are methods of treating, managing or lessening the severity ofsymptoms and infections associated with one or more pulmonary diseasesor infections in a subject, including non-CF associated diseases, themethod comprising administering to the subject a bismuth-thiol (BT)composition that comprises at least one BT compound. In someembodiments, the subject has at least one pulmonary infection, such as aCF-related pulmonary infection. In other embodiments, the subject has atleast two pulmonary infections and the infections are either concurrentor successive in order. The pulmonary infections could be cause by thesame microbial pathogen and be located in two different lungs, or lobesof the lung. In other embodiments, the pulmonary infections could becaused by different microbial pathogens and be located in the same lung,or lobe of the lung. In some embodiments, the pulmonary infection is inone lung, while in others it is present in both lungs. In certainembodiments, the pulmonary infection is in one or more of the threelobes of the right lung. In other embodiments, the pulmonary infectionis in one or both of the two lobes of the left lung. Any combination ofone or more microbial pathogens, microbial pathogen quantity, andinfection location in the lung is contemplated within the term“pulmonary infection”. In some embodiments, the pulmonary infection is abronchiectasis infection, pneumonia, valley fever, allergicbronchopulmonary aspergillosis (ABPA), ventilator acquired pneumonia,hospital acquired pneumonia, community acquired pneumonia, ventilatorassociated tracheobronchitis, lower respiratory tract infection,non-tuberculous Mycobacteria, anthrax, legionellosis, pertussis,bronchitis, Bronchiolitis, COPD-associated infection, and post-lungtransplantation. In some embodiments, the pulmonary infection is abronchiectasis infection.

In some embodiments, the pulmonary infection contains one or morebacterial or fungal pathogens. In some embodiments, the disclosedmethods comprise treating the CF-related pulmonary infection. In someembodiments, the disclosed methods comprise managing the CF-relatedpulmonary infection. In some embodiments, the disclosed methods compriselessening the severity of the CF-related pulmonary infection.

In some embodiments, the methods of the present invention may includetreating, managing or lessening the severity of symptoms and infectionsassociated with one or more pulmonary diseases or infections in asubject by administering to the subject a bismuth-thiol (BT) compositionthat comprises at least one BT compound. In a specific embodiment, thecompound is bismuth-1,2-ethanedithiol (BisEDT).

In certain embodiments, the pulmonary infection is located in or on thelung mucosa, the bronchi and/or the bronchioles. In other embodiments,the pulmonary infection is located on the surface of or within abacterial biofilm, aggregated bacteria, a fungal biofilm, or aggregatedfungi. In some embodiments, the pulmonary infection is located in thesputum wherein the pulmonary infection involves and is, at least inpart, present in the mucous/sputum layers associated with the lungs. Incertain embodiments, the bacterial pathogen comprises one or more ofgram-positive bacteria and gram-negative bacteria. The bacterialpathogen can comprise one or more of a bacterial biofilm and planktonicbacteria. In some embodiments, the fungal pathogen comprises one or moreof a fungal biofilm and planktonic fungi. In certain embodiments, thefungal pathogen is Candida albicans, drug resistant Candida albicans,Candida glabrata, Candida krusei, Candida guilliermondii, Candida auris,Candida tropicalis, Aspergillus niger, Aspergillus terreus, Aspergillusfumigatus, and/or Aspergillus flavus.

In some embodiments, the method comprises at least one of: (i) reducingthe microbial (e.g. bacterial or fungal) biofilm, (ii) impairing growthof the microbial (e.g. bacterial or fungal) biofilm, and (iii)preventing reformation of the microbial (e.g. bacterial or fungal)biofilm. In other embodiments, the BT composition treats, manages orlessens the severity of the pulmonary infection by one or more of:

prevention of the infection by the bacterial or fungalpathogen;—prevention of elaboration or secretion of exotoxins from thebacterial or fungal pathogen;

reduction of the bacterial or fungal pathogen (e.g. as measure by amountor titer);

inhibition of cell viability or cell growth of planktonic cells (e.g.substantially all of the cells) of the bacterial or fungal pathogen;

inhibition of biofilm formation by the bacterial or fungal pathogen;

inhibition of biofilm viability or biofilm growth of biofilm-form cells(e.g. substantially all of the cells) of the bacterial or fungalpathogen; and

reducing the viscosity of the sputum.

In some embodiments, the bismuth-thiol composition comprises a pluralityof microparticles that comprise a bismuth-thiol (BT) compound,substantially all of said microparticles having a volumetric meandiameter of from about 0.4 μm to about 5 μm, and wherein the BT compoundcomprises bismuth or a bismuth salt and a thiol-containing compound. Insome embodiments, the bismuth salt is bismuth nitrate, bismuthsubnitrate, or bismuth chloride. In some embodiments, thethiol-containing compound comprises one or more agents selected from1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione,dithioerythritol, 3,4 dimercaptotoluene, 2,3-butanedithiol,1,3-propanedithiol, 2-hydroxypropanethiol, 1-mercapto-2-propanol,dithioerythritol, dithiothreitol, cysteamine, and alpha-lipoic acid. Insome embodiments, at least 60%, 65%, 70, 75%, 80%, 90%, or 95% of themicroparticles have a volumetric mean diameter of from about 0.4 μm toabout 3 μm, or from about 0.5 μm to about 2 μm, or from about 0.7 μm toabout 2 μm, or from about 0.8 μm to about 1.8 μm, or from about 0.8 μmto about 1.6 μm, or from about 0.9 μm to about 1.4 μm, or from about 1.0μm to about 2.0 μm, or from about 1.0 μm to about 1.8 μm, or any narrowranges between the specific ranges described above.

In some embodiments of the presently disclosed methods, at least 60%,65%, 70, 75%, 80%, 90%, or 95% of the microparticles have a volumetricmean diameter of from about 0.6 μm to about 2.5 μm. In some embodiments,substantially all of the microparticles have a VMD of from about 0.6 μmto about 2.5 μm. In some embodiments, at least 70% of the aerosolizedparticles have a MMAD of about 0.9 μm to about 3 μm. In someembodiments, the composition is a suspension of microparticles having avolumetric mean diameter (VMD) from about 0.6 μm to about 2.5 μm and/ora mass median aerodynamic diameter (MMAD) from about 0.9 μm to about 3μm. In some embodiments, the bismuth-thiol composition comprises aplurality of microparticles that comprise a bismuth-thiol (BT) compound,substantially all of said microparticles having a volumetric meandiameter of from about 0.4 μm to about 5 μm, and wherein the BT compoundcomprises bismuth or a bismuth salt and a thiol-containing compound.

In some embodiments, the BT composition comprises one or more BTcompounds selected from

bismuth-2,3-dimercaptopropanol (2:3 molar ratio, BisBAL)bismuth-dithioerythritol (2:3 molar ratio, BisERY)bismuth-4-methyl-1,2-benzenedithiol (2:3 molar ratio, BisTOL)bismuth-2,3-butanedithiol (BisBDT) bismuth-2,3-butanedithiol,2-mercaptopyridine N-oxide (2:1:2 molar ratio, BisBDT/PYR)bismuth-2,3-dimercaptopropanol, 2-mercaptopyridine N-oxide (2:1:2 molarratio, BisBAL/PYR) bismuth-1,2-ethanedithiol, 2-mercaptopyridine N-oxide(2:1:2 molar ratio, BisEDT/PYR) bismuth-4-methyl-1,2-benzenedithiol,2-mercaptopyridine N-oxide (2:1:2 molar ratio, BisTOL/PYR)bismuth-1,3-propanedithiol, 2-mercaptopyridine N-oxide (2:1:2 molarratio, BisPDT/PYR) bismuth-dithioerythritol, 2-mercaptopyridine N-oxide(2:1:2 molar ratio, BisERY/PYR) bismuth-1-mercapto-2-propanol,1,2-ethanedithiol (1:1:1 molar ratio, BisHPT/EDT) bismuth withethanedithiol and 2-mercaptobenzoimidazole (BisEDT/2MBI (1:1)) bismuthwith ethanedithiol and 2-mercaptopyrimidine (BisEDT/SPN (2MPMD) (1:1))bismuth with ethanedithiol and 3-mercapto-1,2,4-triazole (BisEDT/3MTZ(1:1)) bismuth with ethanedithiol and 1-propane thiol (BisEDT/PT (1:1))bismuth with ethanedithiol and cysteamine (BisEDT/CSTMN (1:1)) bismuthwith ethanedithiol and 3-mercaptopropionic acid (BisEDT/3MPA (1:1))bismuth with lipoic acid (reduced) (BisALA (BisLipo) (1:1.5)) bismuthwith 2-mercaptolpyridine N-oxide and 2-mercaptobenzoimidazole(BisPYR/2MBI (1:1)) bismuth with 2-mercaptolpyridine N-oxide andcysteamine (BisPYR/CSTMN (1:1)) bismuth with 2,3-dimercapto-1-propanoland 2-mercaptobenzoimidazole (BisBAL/2MBI (1:1)) bismuth with2,3-dimercapto-1-propanol and cysteamine (BisBAL/CSTMN (1:1)) bismuthwith 3,4 dimercapto toluene and 2-mercaptobenzoimidazole (BisTOL/2MBI(1:1)) bismuth with 3,4 dimercapto toluene and cysteamine (BisTOL/CSTMN(1:1)) bismuth with 2-mercapto pyridine (BisEDT/MPYR)

In some embodiments, the BT composition comprises one or more BTcompounds selected from Bis-BAL, BisEDT, Bis-dimercaprol, Bis-DTT,Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT,Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bi-sPyr/EDT, Bis-Pyr/PDT,Bis-Pyr/Tol, Bis-Pyr/Ery, bismuth-1-mercapto-2-propanol, andBisEDT/2-hydroxy-1-propanethiol. In other embodiments, the BT compoundis selected from one or more of BisEDT, Bis-Bal, Bis-Pyr, Bis-Ery,Bis-Tol, Bis-BDT, or BisEDT/2-hydroxy-1-propane thiol. As used herein,MB-1B3 (or MB-1-B3) refers to BisEDT; MB-6 refers to BisBDT; MB-8-2refers to BisBDT/PYR; and MB-11 refers to BisEDT/PYR.

In some embodiments, the bismuth thiol compound is BisEDT, which has thefollowing structure:

Combination Treatments

In certain embodiments, compounds disclosed herein can be used alone orconjointly administered with another type of therapeutic agent. As usedherein, the phrase “conjoint administration” refers to any form ofadministration of two or more different therapeutic compounds such thatthe second compound is administered while the previously administeredtherapeutic compound is still effective in the body (e.g., the twocompounds are simultaneously effective in the subject). For example, thedifferent therapeutic compounds can be administered either in the sameformulation or in a separate formulation, either concomitantly orsequentially. In certain embodiments, the different therapeuticcompounds can be administered within one hour, 12 hours, 24 hours, 36hours, 48 hours, 72 hours, or a week of one another. Thus, a subject whoreceives such treatment can benefit from a combined effect of differenttherapeutic compounds.

In certain embodiments, conjoint administration of compounds of thedisclosure with one or more additional therapeutic agent(s) providesimproved efficacy relative to each individual administration of thecompound of the disclosure or the one or more additional therapeuticagent(s). In certain such embodiments, the conjoint administrationprovides an additive effect or synergistic effect, wherein an additiveeffect refers to the sum of each of the effects of individualadministration of the compound of the disclosure and the one or moreadditional therapeutic agent(s). In some embodiments, the subjectreceives conjoint administration of a therapy for another disease,disorder, or condition. In some embodiments, the other therapy is a CFTRmodulator or bronchodilator.

In some embodiments, the methods of the present disclosure comprisecoadministering or conjointly administering to the subject an antibioticselected from amikacin, tobramycin, gentamicin, piperacillin,mezlocillin, ticarcillin, imipenum, ciprofloxacin, ceftazidime,aztreonam, ticaricillin-clavulanate, dicloxacillin, amoxicillin,ticarcillin-clavulanate, trimethoprim-sulfamethoxazole, cephalexin,piperacillin-tazobactam, linezolid, daptomycin, vancomycin,metronidazole, clindamycin, colistin, tetracycline, levofloxacin,amoxicillin and clavulanic acid (Augmentin®), cloxacillin,dicloxacillin, cefdinir, cefprozil, cefaclor, cefuroxime,erythromycin/sulfisoxazole, erythromycin, clarithromycin, azithromycin,doxycycline, minocycline, tigecycline, imipenem, meripenem,colistimethate/colistin®, methicillin, oxacillin, nafcillin,cabenicillin, azlocillin, piperacillin and tazobactam (Zosyn®),cefepime, ethambutol, rifampin, and meropenem. In some embodiments, theantibiotic is selected from meropenem, ceftazidime, tobramycin,amikacin, aztreonam, ciprofloxacin, colistin, and levofloxacin.

In certain embodiments of the present disclosure, the therapeutic agentsthat can be conjointly administered with compounds of the disclosure,such as a bismuth-thiol compound, include known antibiotics. In someembodiments, the antibiotic is selected from methicillin, vancomycin,nafcillin, gentamicin, ampicillin, chloramphenicol, doxycycline,colistin amikacin, aztreonam, and tobramycin. In some embodiments, theantibiotic is selected from tobramycin, imipenem, tetracycline, andminocycline. In some embodiments, the antibiotic is administeredsystemically after revision surgery. In some embodiments, the antibioticis administered prior to revision surgery. The conjointly administeredtherapeutic agent, such as an antibiotic, can be administered with anysuitable frequency and at any suitable dosage. Such dosage amount andfrequency can be determined by those of ordinary skill in the art.

In certain embodiments, BT compounds of the disclosure can be conjointlyadministered with one or more other BT compounds of the disclosure.Moreover, such combinations can be conjointly administered with othertherapeutic agents.

Pharmaceutical Compositions

The compositions and methods of the present disclosure can be utilizedto treat a subject in need thereof. In certain embodiments, the subjectis a mammal such as a human, or a non-human mammal. When administered tosubject, such as a human, the composition or the compound is preferablyadministered as a pharmaceutical composition comprising, for example, acompound of the disclosure and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are well known in the art andinclude, for example, aqueous solutions such as water, physiologicallybuffered saline, physiologically buffered phosphate, or other solventsor vehicles such as glycols, glycerol, oils such as olive oil, orinjectable organic esters. In some embodiments, when such pharmaceuticalcompositions are for human administration, the aqueous solution ispyrogen-free, or substantially pyrogen-free. The excipients can bechosen, for example, to effect delayed release of an agent or toselectively target one or more cells, tissues or organs. Thepharmaceutical composition can be in dosage unit form such as lyophilefor reconstitution, powder, solution, syrup, injection or the like. Thecomposition can also be present in a solution suitable for topicaladministration.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acompound of the disclosure. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose, ordextrans; antioxidants, such as ascorbic acid or glutathione; chelatingagents; low molecular weight proteins; salts; or other stabilizers orexcipients. The choice of a pharmaceutically acceptable carrier,including a physiologically acceptable agent, depends, for example, onthe route of administration of the composition. The preparation orpharmaceutical composition can be a self-emulsifying drug deliverysystem or a self-microemulsifying drug delivery system. Thepharmaceutical composition (preparation) also can be a liposome or otherpolymer matrix, which can have incorporated therein, for example, acompound of the disclosure. Liposomes, for example, which comprisephospholipids or other lipids, are nontoxic, physiologically acceptableand metabolizable carriers that are relatively simple to make andadminister.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of a subject without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols andsugar alcohols, such as glycerin, sorbitol, mannitol, xylitol,erythritol, and polyethylene glycol; (12) esters, such as ethyl oleateand ethyl laurate; (13) agar; (14) buffering agents, such as magnesiumhydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-freewater; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol;(20) phosphate buffer solutions; and (21) other non-toxic compatiblesubstances, including salts such as sodium chloride, employed inpharmaceutical formulations.

The formulations can conveniently be presented in unit dosage form andcan be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, the particular mode of administration. The amountof active ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect.

In some embodiments, the BT composition is a powder, spray, ointment,paste, cream, lotion, solution, patch, suspension or gel. In someembodiments, the BT composition is a solution. The BT composition cancomprise any suitable concentration of bismuth-thiol compound. In someembodiments, the BT composition is administered as a dosage from about0.25 mg/mL to about 15 mg/mL, from about 0.4 mg/mL to about 15 mg/mL,from about 0.6 mg/mL to about 15 mg/mL, from about 0.6 mg/mL to about100 mg/mL, from about 5 mg/mL to about 100 mg/mL, from about 10 mg/mL toabout 100 mg/mL, from about 25 mg/mL to about 100 mg/mL, from about 50mg/mL to about 100 mg/mL, from about 0.8 mg/mL to about 15 mg/mL, fromabout 1 mg/mL to about 10 mg/mL, from 2.5 mg/mL to about 10 mg/mL, fromabout 4 mg/mL to about 10 mg/mL, from about 5 mg/mL to about 10 mg/mL,from about 6 mg/mL to about 10 mg/mL, 0.6 mg/mL to about 6 mg/mL, fromabout 4 mg/mL to about 15 mg/mL, from about 6 mg/mL to about 15 mg/mL,from about 50 μg/mL to about 750 μg/mL, from about 75 μg/mL to about 500μg/mL, from about 100 μg/mL to about 250 μg/mL, from about 100 μg/mL toabout 150 μg/mL, or from about 75 μg/mL to about 150 μg/mL; and/or thetotal amount of the BT composition administered to the lungs is fromabout 0.25 mg to about 15 mg, from about 0.4 mg to about 15 mg, fromabout 0.6 mg to about 15 mg, from about 0.8 mg to about 15 mg, fromabout 1 mg to about 10 mg, from 2.5 mg to about 10 mg, from about 4 mgto about 10 mg, from about 5 mg to about 10 mg, from about 6 mg to about10 mg, 0.6 mg to about 6 mg, from about 4 mg to about 15 mg, from about6 mg to about 15 mg, from about 50 μg to about 750 μg, from about 75 μgto about 500 μg, from about 100 μg to about 250 μg, from about 100 μg toabout 150 μg, or from about 75 μg to about 150 μg. In certainembodiments, the BT composition is administered as a dosage from about0.6 mg/mL to about 6 mg/mL.

In some embodiments, the BT composition is administered three times perday, two times per day, once daily, every other day, once every threedays, once every week, once every other week, once monthly, to onceevery other month. In certain embodiments, the BT composition isadministered once daily. In certain embodiments, the BT composition isadministered once weekly. In certain embodiments, the BT composition isadministered once every other week. In some embodiments, the BTcomposition is administered chronically in a 4 week on/4 week off dosingschedule. In some embodiments, the BT composition is administeredchronically, for example as part of a background therapy. As will beappreciated by a person having ordinary skill in the art, theadministration frequency may depend on a number of factors includingdose and administration route. For example, if the BT composition isadministered via an aerosol administration, a low dose such as 100-1000μg/mL may be administered once or twice daily; however, a high dose suchas 2.5-10 mg/mL may be administered e.g. once or twice a week.

In some embodiments, the BT composition further comprises one or morecarriers selected from animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, polymers, talc, and zincoxide. In some embodiments, the carrier is methylcellulose. In someembodiments, the carrier is poly(methyl methacrylate).

Compositions can also be formulated so as to provide slow or controlledrelease of the active ingredient therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile, other polymer matrices, liposomes and/ormicrospheres. They can be sterilized by, for example, filtration througha bacteria-retaining filter, by ionizing radiation (gamma photons forexample), autoclaving, or by incorporating sterilizing agents in theform of sterile solid compositions that can be dissolved in sterilewater, or some other sterile injectable medium immediately before use.

Liquid dosage forms useful for topical administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,microemulsions, solutions, suspensions, gels, syrups and elixirs. Inaddition to the active ingredient, the liquid dosage forms can containinert diluents commonly used in the art, such as, for example, water orother solvents, cyclodextrins and derivatives thereof, solubilizingagents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, oils (such as cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Besides inert diluents, the topical compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, and preservative agents.

Suspensions, in addition to the active compounds, can contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound can be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives or buffers that can be required.

The ointments, pastes, creams and gels can contain, in addition to anactive compound, one or more excipients or carriers, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc, polymers, salts, and zinc oxide, or mixtures thereof. In someembodiments, the BT composition is in the form of an aqueous solution.In some embodiments, the excipient comprises a salt selected from sodiumchloride or potassium chloride. In some embodiments, the excipientcomprises sodium chloride.

In certain embodiments, the BT composition is a suspension of one ormore BT compounds in TWEEN® (e.g. TWEEN 80®) and/or in a buffer (e.g.sodium phosphate buffer). For example, in some embodiments, the BTcomposition is a suspension of one or more BT compounds in from about0.1% TWEEN 80® to about 1.0% TWEEN 80®, including all rangestherebetween. For example, the BT composition is a suspension of one ormore BT compounds in about 0.1% TWEEN 80®, about 0.2% TWEEN 80®, about0.3% TWEEN 80®, about 0.4% TWEEN 80®, about 0.5% TWEEN 80®, about 0.6%TWEEN 80®, about 0.7% TWEEN 80®, about 0.8% TWEEN 80®, about 0.9% TWEEN80®, or about 1% TWEEN 80®. In some embodiments, the BT composition is asuspension of one or more BT compounds in about 0.5% TWEEN 80®.

In a specific embodiment, the present invention may be a pharmaceuticalcomposition comprising bismuth-thiol (BT) composition that comprisesBisEDT suspended therein, wherein the BT composition comprises aplurality of microparticles. In a specific embodiment, the D90 of saidmicroparticles is less than or equal to 4.5 μm, or 4.0 μm, or 3.5 μm, or3.0 μm, or 2.5 μm, or 2.0 μm, or 1.9 μm, or 1.8 μm, or μm 1.7 μm, or 1.6μm, or 1.5 μm or any ranges in between. In a specific embodiment, theD90 of said microparticles is less than or equal to 1.9 μm. In anotherspecific embodiment, the D90 of said microparticles is less than orequal to 1.6 μm. In another specific embodiment, the D50 of saidmicroparticles is less than or equal to 2.5 μm, or 2.0 μm, or 1.5 μm, or1.3 μm, or 1.2 μm, or 1.1 μm, or 1.0 μm, or 0.9 μm, or 0.87 μm, or 0.72μm or any ranges in between. In another specific embodiment, the D10 ofsaid microparticles is less than or equal to 0.9 μm, or 0.8 μm, or 0.7μm, or 0.6 μm, or 0.50 μm, or 0.40 μm, or 0.39 μm, or 0.38 μm, or 0.37μm, or 0.36 μm, or 0.35 μm, or 0.34 μm, or 0.33 μm, or any ranges inbetween. In a specific embodiment, the pharmaceutical compositioncomprising bismuth-thiol (BT) composition comprises BisEDT suspendedtherein, wherein the BT composition comprises a plurality ofmicroparticles, wherein the D90 of said microparticles is less than orequal to about 1.6 μm. In a specific embodiment, the BT compositioncomprises BisEDT at a concentration greater than about 0.1 mg/mL, about0.05% to about 1.0% TWEEN 80®, about 0.05 to 40 mM sodium chloride, andoptionally about 2 to 20 mM sodium phosphate at about pH. 7.4. Inanother specific embodiment, the compositions described above can beadministered to a subject for treating, managing and/or lessening theseverity of cystic fibrosis (CF) symptoms and infections in saidsubject, or any specific method of treating, managing and/or lesseningthe severity of cystic fibrosis (CF) symptoms described herein. Inanother specific embodiment, the compositions described above can beadministered to a subject for treating, managing and/or lessening theseverity of symptoms and infections associated with one or morepulmonary diseases or infections in a subject or any specific method oftreating, managing and/or lessening the severity of symptoms andinfections associated with one or more pulmonary diseases describedherein.

A variety of buffers may be used in the context of the presentdisclosure and will be readily apparent to a person having ordinaryskill in the art. For example, in some embodiments, suitable buffersinclude sodium or potassium citrate, citric acid, phosphate buffers suchas sodium phosphate, boric acid, sodium bicarbonate and various mixedphosphate buffers including combinations of Na₂HPO₄, NaH₂PO₄ and KH₂PO₄.In some embodiments, sodium phosphate buffer is used. In someembodiments, sodium citrate buffer is used. Without being bound by anyparticular theory, changes in airway surface liquid pH may contribute tothe host defense defect in cystic fibrosis soon after birth. Changes inlung pH may impact the airway surface liquid environment, improve airwaydefenses, and alter the disease course. Accordingly, the formulation pHmay vary from about 5 to about 10. In some embodiments, the formulationpH is about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10. In someembodiments, the formulation pH is about 7.4.

In some embodiments, the BT composition is a suspension of one or moreBT compounds in about 0.5% TWEEN 80®in sodium phosphate buffer at a pHof about 7.4. In some embodiments, the one or more BT compounds arepresent in the composition at a concentration ranging from about 100μg/mL to about 1000 mg/mL including all integers and rangestherebetween. For example, in some embodiments, the one or more BTcompounds are present in the composition at a concentration ranging fromabout 100 μg/mL, 200 μg/mL, 300 μg/mL, 400 μg/mL, 500 μg/mL, 600 μg/mL,700 μg/mL, 800 μg/mL, 900 μg/mL, 1000 μg/mL, 10 mg/mL, 25 mg/mL, 50mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, 225 mg/mL,250 mg/mL, 275 mg/mL, 300 mg/mL, 325 mg/mL, 350 mg/mL, 375 mg/mL, 400mg/mL, 425 mg/mL, 450 mg/mL, 475 mg/mL, 500 mg/mL, 525 mg/mL, 550 mg/mL,575 mg/mL, 600 mg/mL, 625 mg/mL, 650 mg/mL, 675 mg/mL, 700 mg/mL, 725mg/mL, 750 mg/mL, 775 mg/mL, 800 mg/mL, 825 mg/mL, 850 mg/mL, 875 mg/mL,900 mg/mL, 925 mg/mL, 950 mg/mL, 975 mg/mL, to about 1000 mg/mL. In someembodiments, the one or more BT compounds are present in the compositionat a concentration ranging from about 100 μg/mL to about 1000 μg/mL.

In some embodiments, the composition osmolality may need to be furtheradjusted with an additive such as NaCl or TDAPS to achieve a desiredosmolality. For example, in some embodiments, the osmolality of thecomposition is adjusted with sodium chloride to an osmolality rangingfrom about 100 mOsmol/kg to about 500 mOsmol/kg, including all integersand ranges therebetween. In some embodiments, the osmolality of thecomposition is from about 290 mOsmol/kg to about 310 mOsmol/kg. Forexample, in some embodiments, the osmolality of the composition is about290 mOsmol/kg, 291 mOsmol/kg, 292 mOsmol/kg, 293 mOsmol/kg, 294mOsmol/kg, 295 mOsmol/kg, 296 mOsmol/kg, 297 mOsmol/kg, 298 mOsmol/kg,299 mOsmol/kg, 300 mOsmol/kg, 301 mOsmol/kg, 302 mOsmol/kg, 303mOsmol/kg, 304 mOsmol/kg, 305 mOsmol/kg, 306 mOsmol/kg, 307 mOsmol/kg,308 mOsmol/kg, 309 mOsmol/kg, to about 310 mOsmol/kg. In someembodiments, the osmolality is about 300 mOsmol/kg.

In some embodiments, the BT composition is a suspension of BisEDT inTWEEN® (e.g. TWEEN 80®) in a buffer (e.g. sodium phosphate buffer). Insome embodiments, the BT composition is a suspension of BisEDT in about0.5% TWEEN 80® in a sodium phosphate buffer at a pH of about 7.4. Insome embodiments, the BT composition is a suspension of BisEDT in about0.5% TWEEN 80® in a sodium phosphate buffer at a pH of about 7.4,wherein the composition has an osmolality of about 300 mOsmol/kg (e.g.adjusted to 300 mOsmol/kg with sodium chloride). In some embodiments,the BisEDT is present at a concentration of about 100 μg/mL, 250 μg/mL,500 μg/mL, 750 μg/mL, 1000 μg/mL, 2.5 mg/mL, 10 mg/mL, 25 mg/mL, 50mg/mL, 75 mg/mL, or about 100 mg/mL.

In some embodiments, the BT composition is a suspension formulationwhich is intended for pulmonary delivery. For example, the BTcomposition is a suspension formulation which is ultimately administeredby inhalation either orally and/or nasally. Accordingly, in someembodiments, the BT composition is aerosolized by a device such as anebulized.

Powders and sprays can contain, in addition to an active compound,excipients such as methylcellulose, sodium chloride, PMMA, lactose,talc, silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, dipalmitoylphosphatidylcholine (DPPC), leucine,polyethyleneglycol, or mixtures of these substances. Sprays canadditionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

In some embodiments, the BT composition is administered by inhalation,orally or nasally, using an aerosol device, such as a nebulizer. Knownnebulizers, such as PARI LC PLUS®, can administer the disclosedcompositions as an aqueous solution, optionally in buffered saline. Thesolution can be provided to the subject in the form of an ampule for usein the nebulizer. The nebulizer can be reusable and includes acompressor that provides the formulation over a period of time, such asabout 10-15 minutes or longer. Known compressors, such as APRI Vios Airand DeVilbiss Pulmo-aide, are suitable for administration. The nebulizeradministers the formulation topically to the lung tissues, such asmucosa, the bronchi and/or the bronchioles, alveoli, deep lung alveoli.The formulation can penetrate lung mucosa and biofilms to reduce themicrobial (e.g. bacterial or fungal) biofilm, impair the growth of themicrobial (e.g. bacterial or fungal) biofilm, prevent reformation of themicrobial (e.g. bacterial or fungal) biofilm, reduce planktonic growth,and/or inhibit planktonic growth.

In other embodiments, a nose-only aerosol device can be used foradministration of the formulation.

An exemplary BT composition formulation is a neutral pH, isotonic,buffered aqueous solution of BT compound microparticles with a nonionicsurfactant. In certain embodiments, the buffer is a phosphate bufferwith added NaCl. In some embodiments, the microparticle size is a D₅₀ ofabout 1-5 μm. The formulation can be delivered using commerciallyavailable compressed air jet nebulizer. In some embodiments, theformulation concentration is about 0.1 μg/mL to about 100 mg/mL.

In some embodiments, the present disclosure provides an aerosolcomprising a plurality of dispersed liquid droplets in a gas, saidliquid droplets comprising a BT composition comprising at least one BTcompound suspended therein, wherein the BT compound comprises bismuthand/or a bismuth salt and a thiol-containing compound; and wherein atleast 60%, 65%, 70, 75%, 80%, 90%, or 95% of the liquid droplets have amass median aerodynamic diameter (MMAD) from about 0.4 μm to about 5μmwhen measured by laser time of flight and/or cascade impactor. In someembodiments, at least 60%, 65%, 70, 75%, 80%, 90%, or 95% of the liquiddroplets have a MMAD of from about 0.4 μm to about 7 μm, or from about0.5 μm to about 5 μm, or from about 0.7 μm to about 4 μm, or from about0.7 μm to about 3.5 μm, or from about 0.8 μm to about 3.5 μm, or fromabout 0.9 μm to about 3.5 μm, or from about 0.9 μm to about 3 μm, orfrom about 0.8 μm to about 1.8 μm, or from about 0.8 μm to about 1.6 μm,or from about 0.9 μm to about 1.4 μm, or from about 1.0 μm to about 2.0μm, or from about 1.0 μm to about 1.8 μm, including all rangestherebetween. In some embodiments, at least 60%, 65%, 70, 75%, 80%, 90%,or 95% of the liquid droplets have a MMAD of from about 0.8 μm to about1.6 μm, or from about 0.9 μm to about 3.5 μm, or from about 0.9 μm toabout 3 μm, or from about 0.9 μm to about 1.4 μm, or from about 1.0 μmto about 2.0 μm, or from about 1.0 μm to about 1.8 μm, and all rangestherebetween.

In some embodiments, the plurality of liquid droplets have a D90 of lessthan about 10 μm. For example, in some embodiments, the plurality ofliquid droplets have a D90 of less than about 10 μm, 9 μm, 8 μm, 7 μm, 6μm, 5 μm, 4 μm, 3 μm, 2 μm, or about 1 μm. In some embodiments, theplurality of liquid droplets have a D90 of less than about 3 μm. In someembodiments, the plurality of liquid droplets have a D90 ranging fromabout 1 μm to about 5 μm, or about 2 μm to about 6 μm, or about 2 μm toabout 4 μm, or about 2 μm to about 3 μm, or about 1 μm to about 4 μm, orabout 1 μm to about 3 μm.

In some embodiments, the plurality of liquid droplets are dispersed in acontinuous gas phase.

In some embodiments, the BT compound of the aerosol comprises bismuthand/or a bismuth salt associated covalently and/or in a coordinationcomplex with one or more thiol-containing compounds. For example, thebismuth salt is bismuth nitrate, bismuth subnitrate, or bismuthchloride. In some embodiments, the thiol-containing compound comprisesone or more agents selected from 1,2-ethane dithiol,2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4dimercaptotoluene, 2,3-butanedithiol, 1 ,3-propanedithiol,2-hydroxypropanethiol, 1-mercapto-2-propanol, dithioerythritol,dithiothreitol and alpha-lipoic acid. In some embodiments, the BTcomposition comprises one or more BT compounds selected from BisBAL,BisEDT, Bis-dimercaprol, BisDTT, Bis-2-mercaptoethanol, Bis-DTE,Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT,BisPyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,bismuth-1-mercapto-2-propanol, BisEDT/CSTMN (1:1), BisPYR/CSTMN (1:1),BisBAL/CSTMN (1:1), BisTOL/CSTMN (1:1), andBisEDT/2-hydroxy-1-propanethiol. In some embodiments, the BT compound isselected from one or more of BisEDT, Bis-Bal, Bis-Pyr, Bis-Ery, Bis-Tol,Bis-BDT, or BisEDT/2-hydroxy-1-propane thiol. In some embodiments, theBT compound on the aerosol is BisEDT. In some embodiments, the BTcompound of the aerosol is BisBDT or BisBAL.

In some embodiments, the BT compound (e.g. BisEDT) is suspended in theliquid droplet. The BT compounds of the present disclosure have littleto no solubility in conventional solvents and aerosol carriers andtherefore exist substantially as a suspension of BT particles in theaerosol droplet. For example, in some embodiments, the BT compound (suchas BisEDT) is less than 1% soluble in the aerosol carrier and thereforeexists primarily (>99%) as a solid.

In some embodiments, the droplets further comprise TWEEN 80™ (e.g. fromabout 0.05% to about 1%) and optionally a buffer (e.g. sodium phosphateor sodium citrate) at a pH of about 7.4; and/or sodium chloride.

The aerosols of the present disclosure have a very narrow MMADdistribution which is beneficial because of the need to concentrate theparticle mass in the target size range, and minimize or eliminate thefraction of the product that is outside of the respirable range or‘fines’, i.e. particles of typically less than 0.4 μm diameter. Theability to create a narrow droplet size distribution in the appropriatesize range provides control of the initial evaporation rate and allowsfor high deposition efficiency. The limiting factor in terms of thelower limit of particle aerosol droplet size is the BT microparticlesize (e.g. the BisEDT microparticle size). An aerosolized droplet cannotbe smaller than the BisEDT microparticulate size. As such, the BTmicroparticle size distribution, as well as the uniformity andconsistent reproducibility of the BT microparticulate size distribution,are important beneficial characteristics to support the generation of asafe, effective, and efficient aerosolized BisEDT drug product forinhalation purposes. Accordingly, in some embodiments, the aerosols ofthe present disclosure effectuate a deposition efficiency of greaterthan 3%, greater than 5%, greater than 10%, greater than 15%, greaterthan 20%, greater than 25%, greater than 30%, greater than 35%m greaterthan 40%, greater than 45%, greater than 50%, greater than 55%, greaterthan 60%, greater than 65%, greater than 70%, greater than 75%, andgreater than 80%. In some embodiments, the deposition efficiency refersto deposition to the deep lung region of lung, for example, to the deeplung alveoli. In some embodiments, the aerosols of the presentdisclosure effectuate a deposition efficiency upon aerosolization via anebulizer. For example, the nebulizer is a jet nebulizer. In someembodiments, the jet nebulizer is a PARI LC PLUS® jet nebulizer or PARILC SPRINT® jet nebulizer. In some embodiments, the nebulizer has aninlet pressure from about 10 to about 40 psig (e.g. 20-25 psig). In someembodiments, the inlet flow is from about 3 L/min to about 8 L/min (e.g.5.2 L/min). In some embodiments, the exhaust air flow is from about 3L/min to about 8 L/min (e.g. 5 L/min).

The alveolar region of the lung has a minimal thickness (0.5 μm-2.5 μm)separating the blood flow from the lumen so conventional pulmonaryagents that deposit on the alveolar epithelium have extremely short lungresidence time due to systemic absorption. Accordingly, conventionalpulmonary treatments typically require frequent dosing in order tomaintain adequate levels of drug at the tissue level. However, theaerosolized particles of the present disclosure were surprisinglydiscovered to possess an exceptionally long residence time in the lungs(measured as half-life) and have reduced mucociliary clearance andmacrophage uptake relative to conventional pulmonary treatments.Furthermore, the long residence time of the aerosols of the presentminimizes systemic activity and associated systemic side effects.Without being bound by any particular theory, it is believed that theaerosolized microparticles dissolve slowly on the lung lumen and thesystemic exposure is thus dissolution rate limited. Further, theincreased lung residence time results in significant reductions inmicrobial colony due to the continuous presence of the BTmicroparticles.

Accordingly, in some embodiments, when the aerosol is deposited to thelung (e.g. to the deep lung alveoli), the BT compounds have an averagehalf-life of at least 2 days. For example, the BT compounds have anaverage half-life of about 2, 3, 4, or 5 days. In some embodiments, theBT compound is BisEDT. In a specific embodiment, the lung tissuehalf-life of BisEDT is 30 hrs or more, 40 hrs or more, 50 hrs or more,60 hrs or more, 70, hrs or more, 80 hrs or more, 90 hrs or more, 100 hrsor more, 110 hrs or more, 125 hrs or more, or 150 hrs or more. In aspecific embodiment, the lung tissue half-life is after a single dosevia inhalation. In another specific embodiment, lung tissue is from arat. In another specific embodiment, lung tissue half-life of BisEDT isdetermined by the ue of protocol as in Example 8 herein.

In another embodiment, the lung tissue half-life of BisEDT is 80 hrs ormore when the rat is given a single dose of 100 μg/kg lung using a PARTLC PLUS® jet nebulizer to administer to the rats with the formulationsdescribed herein. In another embodiment, the lung tissue half-life ofBisEDT is 90 hrs or more. In another embodiment, the lung tissuehalf-life of BisEDT is 100 hrs or more.

In another embodiment, after delivering the aerosolized composition to asubject, at least 60%, 65%, 70, 75%, 80%, 90%, or 95% of the dose isdeposited on the lung, as opposed to the orpharanygeal region and theconducting airways. In a specific embodiment, at least 80% of the doseis deposited on the lung, as opposed to the orpharanygeal region and theconducting airways. In another specific embodiment, at least 90% of thedose is deposited on the lung, as opposed to the orpharanygeal regionand the conducting airways.

It was previously unheard of for an aerosolized pulmonary treatment tohave aerosol particles with a narrow distribution that effectuate a highdeposition efficiency coupled with an exceptionally long lung residencetime for continuous treatment and little to no systemic absorption.

In some embodiments, the present disclosure provides a method oftreating, managing or lessening the severity of cystic fibrosis (CF)symptoms and infections in a subject, the method comprisingadministering to the subject a bismuth-thiol (BT) composition thatcomprises at least one BT compound, wherein the composition is asuspension of microparticles having a volumetric mean diameter (VMD)from about 0.4 μm to about 5 μm and/or a mass median aerodynamicdiameter (MMAD) from about 0.4 μm to about 5 μm. In some embodiments,the BT compound is BisEDT. In some embodiments, the BT compositioncomprises BisEDT at a concentration greater than about 0.1 mg/mL, about0.05% to about 1.0% TWEEN 80®, about 0.05 to 40 mM sodium chloride, andoptionally about 2 to 20 mM sodium phosphate at about pH. 7.4. Forexample, in some embodiments, the BT composition comprises BisEDT at aconcentration greater than about 0.25 mg/mL, about 0.5% TWEEN 80®, about10 mM sodium chloride, and about 10 mM sodium phosphate at about pH 7.4.In another embodiment of the methods herein, the BT composition isadministered by aerosolization. In some embodiments, when the aerosol isdeposited to the lung (e.g. to the deep lung alveoli), the BT compoundshave an average half-life of at least 2 days. For example, the BTcompounds have an average half-life of about 2, 3, 4, or 5 days. In someembodiments, the BT compound is BisEDT. In a specific embodiment, thelung tissue half-life of BisEDT is 30 hrs or more, 40 hrs or more, 50hrs or more, 60 hrs or more, 70, hrs or more, 80 hrs or more, 90 hrs ormore, 100 hrs or more, 110 hrs or more, 125 hrs or more, or 150 hrs ormore. In a specific embodiment, the lung tissue half-life is after asingle dose via inhalation. In another specific embodiment, lung tissueis from a rat. In another specific embodiment, lung tissue half-life ofBisEDT is determined by the ue of protocol as in Example 8 herein.

In another embodiment, after delivering the aerosolized composition to asubject, at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% of the dose isdeposited on the lung, as opposed to the orpharanygeal region and theconducting airways. In a specific embodiment, at least 80% of the doseis deposited on the lung, as opposed to the orpharanygeal region and theconducting airways. In another specific embodiment, at least 90% of thedose is deposited on the lung, as opposed to the orpharanygeal regionand the conducting airways. In a specific embodiment, the subject is arat. In another specific embodiement, the percent deposition isdetermined using a PART LC PLUS® jet nebulizer to administer to the ratswith the formulations described herein.

In another embodiment, the lung tissue half-life of BisEDT is 80 hrs ormore when the rat is given a single dose of 100 μg/kg lung using a PARTLC PLUS® jet nebulizer to administer to the rats with the formulationsdescribed herein. In another embodiment, the lung tissue half-life ofBisEDT is 90 hrs or more. In another embodiment, the lung tissuehalf-life of BisEDT is 100 hrs or more.

In another embodiment, the methods of the present invention may includetreating, managing or lessening the severity of cystic fibrosis (CF)symptoms and infections in a subject, by administering to the subject abismuth-thiol (BT) composition that comprises at least one BT compound,wherein the composition is a suspension of microparticles having avolumetric mean diameter (VMD) from about 0.4 μm to about 5 μm and/or amass median aerodynamic diameter (MMAD) from about 0.4 μm to about 5 μm.

In some embodiments, the composition is a suspension of microparticleshaving a volumetric mean diameter (VMD) from about 0.4 μm to about 5 μm.In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% ofthe microparticles have a VMD of from about 0.4 μm to about 5 μm, orfrom about 0.6 μm to about 2.5 μm, or from about 0.7 μm to about 4 μm,or from about 0.7 μm to about 3.5 μm, or from about 0.7 μm to about 3.0μm, or from about 0.9 μm to about 3.5 μm, or from about 0.9 μm to about3 μm, or from about 0.8 μm to about 1.8 μm, or from about 0.8 μm toabout 1.6 μm, or from about 0.9 μm to about 1.4 μm, or from about 1.0 μmto about 2.0 μm, or from about 1.0 μm to about 1.8 μm and all rangestherebetween. In some embodiments, at least 60%, 65%, 70%, 75%, 80%,90%, or 95% of the microparticles have a VMD of from about 0.6μm toabout 2.5μm, or from about 0.8 μm to about 1.6 μm, or from about 0.9 μmto about 3.5 μm, or from about 0.9 μm to about 3μm, or from about 0.9 μmto about 1.4 μm, or from about 1.0 μm to about 2.0 μm, or from about 1.0μm to about 1.8 μm and all ranges therebetween. In some embodiments, themicroparticles have a D90 of less than about 10 μm. For example, in someembodiments, the microparticles have a D90 of less than about 10 μm, 9μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or about 1 μm. In someembodiments, the microparticles have a D90 of less than about 3 μm. Insome embodiments, the microparticles have a D90 ranging from about 1 μmto about 5 μm, or about 2 μm to about 6 μm, or about 2 μm to about 4 μm,or about 2 μm to about 3 μm, or about 1 μm to about 4 μm, or about 1 μmto about 3 μm.

In some embodiments, the BT composition is aerosolized, wherein theaerosolized liquid droplets have a MMAD from about of from about 0.4 μmto about 5 μm. In some embodiments, at least 60%, 65%, 70%, 75%, 80%,90%, or 95% of the liquid droplets have a MMAD of from about 0.4 μm toabout 7 μm, or from about 0.5 μm to about 5 μm, or from about 0.7 μm toabout 4 μm, or from about 0.7 μm to about 3.5 μm, or from about 0.8 μmto about 3.5 μm, or from about 0.9 μm to about 3.5 μm, or from about 0.9μm to about 3 μm, or from about 0.8 μm to about 1.8 μm, or from about0.8 μm to about 1.6 μm, or from about 0.9 μm to about 1.4 μm, or fromabout 1.0 μm to about 2.0 μm, or from about 1.0 μm to about 1.8 μm andall ranges therebetween. In some embodiments, at least 60%, 65%, 70%,75%, 80%, 90%, or 95% of the liquid droplets have a MMAD of from about0.8 μm to about 1.6 μm, or from about 0.9 μm to about 3.5 μm, or fromabout 0.9 μm to about 3 μm, or from about 0.9 μm to about 1.4 μm, orfrom about 1.0 μm to about 2.0 μm, or from about 1.0 μm to about 1.8 μmand all ranges therebetween. In some embodiments, the plurality ofliquid droplets have a D90 of less than about 10 μm. For example, insome embodiments, the plurality of liquid droplets have a D90 of lessthan about 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, orabout 1 μm. In some embodiments, the plurality of liquid droplets have aD90 of less than about 3 μm. In some embodiments, the plurality ofliquid droplets have a D90 ranging from about 1 μm to about 5 μm, orabout 2 μm to about 6 μm, or about 2 μm to about 4 μm, or about 2 μm toabout 3 μm, or about 1 μm to about 4 μm, or about 1 μm to about 3 μm.

In some embodiments, the plurality of liquid droplets are dispersed in acontinuous gas phase.

In some embodiments, the BT compound of the aerosol comprises bismuthand/or a bismuth salt associated covalently and/or in a coordinationcomplex with one or more thiol-containing compounds. For example, thebismuth salt is bismuth nitrate, bismuth subnitrate, or bismuthchloride. In some embodiments, the thiol-containing compound comprisesone or more agents selected from 1,2-ethane dithiol,2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4dimercaptotoluene, 2,3-butanedithiol, 1 ,3-propanedithiol,2-hydroxypropanethiol, 1-mercapto propanol, dithioerythritol,dithiothreitol, cysteamine, and alpha-lipoic acid. In some embodiments,the BT composition comprises one or more BT compounds selected fromBisBAL, BisEDT, Bis-dimercaprol, BisDTT, Bis-2-mercaptoethanol, Bis-DTE,Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT,BisPyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery, BisEDT/CSTMN (1:1),BisPYR/CSTMN (1:1), BisBAL/CSTMN (1:1), BisTOL/CSTMN (1:1),bismuth-1-mercapto-2-propanol, and BisEDT/2-hydroxy-1-propanethiol. Insome embodiments, the BT compound is selected from one or more ofBisEDT, Bis-Bal, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, orBisEDT/2-hydroxy-1-propane thiol. In some embodiments, the BT compoundon the aerosol is BisEDT. In some embodiments, the BT compound of theaerosol is BisBDT or BisBAL.

In some embodiments of the presently disclosed compositions, at least60%, 65%, 70%, 75%, 80%, 90%, or 95% of the microparticles have avolumetric mean diameter of from about 0.6 μm to about 2.5 μm. In someembodiments, substantially all of the microparticles have a VMD of fromabout 0.6 μm to about 2.5 μm. In some embodiments, at least 70% of theaerosolized particles have a MMAD of about 0.9 μm to about 3 μm. In someembodiments, the composition is a suspension of microparticles having avolumetric mean diameter (VMD) from about 0.6 μm to about 2.5 μm and/ora mass median aerodynamic diameter (MMAD) from about 0.9 μm to about 3μm. In some embodiments, the bismuth-thiol composition comprises aplurality of microparticles that comprise a bismuth-thiol (BT) compound,substantially all of said microparticles having a volumetric meandiameter of from about 0.4 μm to about 5 μm, and wherein the BT compoundcomprises bismuth or a bismuth salt and a thiol-containing compound.

In some embodiments, the composition is aerosolized via a nebulizer. Forexample, the nebulizer is a jet nebulizer or vibrating mesh nebulizer.In some embodiments, the jet nebulizer is a PART LC PLUS® jet nebulizeror PARI LC SPRINT® jet nebulizer. In some embodiments, the nebulizer hasan inlet pressure from about 10 to about 40 psig (e.g. 20-25 psig). Insome embodiments, the inlet flow is from about 3 L/min to about 8 L/min(e.g. 5.2 L/min). In some embodiments, the exhaust air flow is fromabout 3 L/min to about 8 L/min (e.g. 5 L/min).

Examples of suitable aqueous and nonaqueous carriers that can beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions can also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agentscan also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form can be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, can depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions can be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the subject being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount can include, but are not limited to, the severityof the subject's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the compound of the disclosure. A larger total dosecan be delivered by multiple administrations of the agent. Methods todetermine efficacy and dosage are known to those skilled in the art(Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13ed., 1814-1882, herein incorporated by reference).

In general, a suitable dose of an active compound used in thecompositions and methods of the disclosure will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

This disclosure includes the use of pharmaceutically acceptable salts ofcompounds of the disclosure in the compositions and methods of thepresent disclosure. In certain embodiments, contemplated salts of thedisclosure include, but are not limited to, alkyl, dialkyl, trialkyl ortetra-alkyl ammonium salts. In certain embodiments, contemplated saltsof the disclosure include, but are not limited to, L-arginine,benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol,diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine,ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium,L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine,potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine,tromethamine, and zinc salts. In certain embodiments, contemplated saltsof the disclosure include, but are not limited to, Na, Ca, K, Mg, Zn orother metal salts.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, and the like. Mixtures of such solvates can also beprepared. The source of such solvate can be from the solvent ofcrystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Embodiments

-   1. A method of treating, managing or lessening the severity of    cystic fibrosis (CF) symptoms and infections in a subject, the    method comprising administering to the subject a bismuth-thiol (BT)    composition that comprises at least one BT compound.-   2. The method of embodiment 1, wherein the subject has at least one    pulmonary infection.-   3. The method of embodiment 2, wherein the subject has at least two    pulmonary infections and the infections are either concurrent or    successive in order.-   4. The method of embodiment 2 or 3, wherein the pulmonary infection    is in one lung.-   5. The method of any one of embodiments 2-4, wherein the pulmonary    infection is in both lungs.-   6. The method of any one of embodiments 2-5, wherein the pulmonary    infection is in one or more of the three lobes of the right lung.-   7. The method of any one of embodiments 2-6, wherein the pulmonary    infection is in one or both of the two lobes of the left lung.-   8. The method of any one of embodiments 2-7, wherein the pulmonary    infection is bronchiectasis infection, pneumonia, valley fever,    allergic bronchopulmonary aspergillosis (ABPA), ventilator acquired    pneumonia, hospital acquired pneumonia, community acquired    pneumonia, ventilator associated tracheobronchitis, lower    respiratory tract infection, non-tuberculous Mycobacteria, anthrax,    legionellosis, pertussis, bronchitis, Bronchiolitis, COPD-associated    infection, and post-lung transplantation.-   9. The method of any one of embodiments 2-8, wherein the pulmonary    infection contains one or more bacterial and/or fungal pathogens.-   10. The method of any one of embodiments 2-9, wherein the pulmonary    infection is a CF-related pulmonary infection.-   11. The method of embodiment 10, comprising treating the CF-related    pulmonary infection.-   12. The method of embodiment 10, comprising managing the CF-related    pulmonary infection.-   13. The method of embodiment 10, comprising lessening the severity    of the CF-related pulmonary infection.-   14. The method of any one of embodiments 2-13, wherein the pulmonary    infection is located in or on the lung mucosa, the bronchi, the    alveoli, the macrophages, and/or the bronchioles.-   15. The method of any one of embodiments 2-13, wherein the pulmonary    infection is located on the surface of or within a bacterial    biofilm, aggregated bacteria, a fungal biofilm, a combined    multispecies or multiphylum biofilm comprised of both bacteria and    fungi, or aggregated fungi.-   16. The method of any one of embodiments 2-13, wherein the pulmonary    infection is located in the sputum.-   17. The method of any one of embodiments 9-16, wherein the bacterial    pathogen comprises one or more of gram-positive bacteria and/or    gram-negative bacteria.-   18. The method of any one of embodiments 9-17, wherein the bacterial    pathogen comprises one or more of a bacterial biofilm and/or    planktonic bacteria.-   19. The method of embodiment 18, wherein the method comprises at    least one of: (i) reducing the bacterial biofilm, (ii) impairing    growth of the bacterial biofilm, (iii) preventing initial formation    of the bacterial biofilm, and/or (iv) preventing reformation of the    bacterial biofilm.-   20. The method of any one of embodiments 9-16, wherein the fungal    pathogen comprises planktonic fungi and/or biofilm fungi.-   21. The method of any one of embodiments 9-20, wherein the BT    composition treats, manages or lessens the severity of the pulmonary    infection by one or both of:

prevention of the infection by the bacterial or fungal pathogen; and

reduction of the bacterial or fungal pathogen; and/or

reducing the viscosity of the sputum.

-   22. The method of any one of embodiments 9-20, wherein the BT    composition treats, manages or lessens the severity of the pulmonary    infection by one or more of:

prevention of elaboration or secretion of exotoxins from the bacterialor fungal pathogen;

inhibition of cell viability or cell growth of planktonic cells of thebacterial or fungal pathogen;

inhibition of biofilm formation by the bacterial or fungal pathogen;

inhibition of biofilm invasiveness to pulmonary tissues;

inhibition of biofilm pathogenicity to pulmonary tissues; and

inhibition of biofilm viability or biofilm growth of biofilm-formingcells of the bacterial or fungal pathogen.

-   23. The method of any one of embodiments 9-22, wherein the one or    more pathogens are selected from Haemophilus influenzae, Pseudomonas    aeruginosa, Staphylococcus aureus, Staphylococcus warneri    Staphylococcus lugdunensis, Staphylococcus epidermidis,    Streptococcus milleri/anginous, Streptococcus pyogenes,    non-tuberculosis Mycobacterium, Mycobacterium tuberculosis,    Burkholderia spp., Achromobacter xylosoxidans, Pandoraea sputorum,    Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, Haemophilus    pittmaniae, Serratia marcescens, Candida albicans, drug resistant    Candida albicans, Candida glabrata, Candida krusei, Candida    guilliermondii, Candida auris, Candida tropicalis, Aspergillus    niger, Aspergillus terreus, Aspergillus fumigatus, Aspergillus    flavus, Morganella morganii, Inquilinus limosus, Ralstonia    mannitolilytica, Pandoraea apista, Pandoraea pnomenusa, Pandoraea    sputorum, Bdellovibrio bacteriovorus, Bordetella bronchiseptica,    Vampirovibrio chlorellavorus, Actinobacter baumanni, Cupriadidus    metallidurans, Cupriavidus pauculus, Cupriavidus respiraculi,    Delftia acidivordans, Exophilia dermatitidis, Herbaspirillum    frisingense, Herbaspirillum seropedicae, Klebsiella pneumoniae,    Pandoraea norimbergensis, Pandoraea pulmonicola, Pseudomonas    mendocina, Pseudomonas pseudoalcaligenes, Pseudomonas putida,    Pseudomonas stutzeri, Ralstonia insidiosa, Ralstonia pickettii,    Neisseria gonorrhoeae, NDM-1 positive E. coli, Enterobacter cloaca,    Vancomycin-resistant E. faecium, Vancomycin-resistant E.    faecalis, E. faecium, E. faecalis, Clindamycin-resistant S.    agalactiae, S. agalactiae, Bacteroides fragilis, Clostridium    difficile, Streptococcus pneumonia, Moraxella catarrhalis,    Haemophilus haemolyticus, Haemophilus parainfluenzae, Chlamydophilia    pneumoniae, Mycoplasma pneumoniae, Atopobium, Sphingomonas,    Saccharibacteria, Leptotrichia, Capnocytophaga, Oribacterium,    Aquabacterium, Lachnoanaerobaculum, Campylobacter, Acinetobacter;    Agrobacterium; Bordetella; Brevundimonas; Chryseobacterium; Delftia;    Enterobacter; Klebsiella; Pandoraea; Pseudomonas; Ralstonia, and    Prevotella.-   24. The method of embodiment 23, wherein the non-tuberculosis    mycobacterium is selected from Mycobacterium abscessus,    Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium    fortuitum, Mycobacterium gordonae, Mycobacterium kansasii,    Mycobacterium avium complex, Mycobacterium marinum, Mycobacterium    terrae and Mycobacterium cheloni.-   25. The method of embodiment 23, wherein the Burkholderia spp. is    selected from Burkholderia cepacia, Burkholderia cepacia complex,    Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia    stabilis, Burkholderia vietnamiensis, Burkholderia dolosa,    Burkholderia ambifaria, Burkholderia anthina, Burkholderia    pyrrocinia, Burkholderia gladioli, Burkholderia ubonensis,    Burkholderia arboris, Burkholderia latens, Burkholderia lata,    Burkholderia metallica, Burkholderia seminalis, Burkholderia    contaminans, and Burkholderia diffusa.-   26. The method of embodiment 23, wherein the one or more pathogens    are selected from Pseudomonas aeruginosa, single drug-resistant    Pseudomonas aeruginosa, multi drug-resistant Pseudomonas aeruginosa,    Staphylococcus aureus, single drug-resistant Staphylococcus aureus,    multi drug-resistant Staphylococcus aureus, methicillin resistant    Staphylococcus aureus, Mycobacterium abscessus, Mycobacterium avium,    Haemophilus influenzae, Burkholderia cepacia, Burkholderia    multivorans, Burkholderia cenocepacia, Burkholderia dolosa,    Achromobacter xylosoxidans, Stenotrophomonas maltophilia,    Staphylococcus epidermidis, and Burkholderia vietnamiensis.-   27. The method of embodiment 23, wherein the one or more pathogens    are selected from Haemophilus influenzae, Pseudomonas aeruginosa,    and Staphylococcus aureus.-   28. The method of any one of embodiments 18, 19 and 22, wherein the    one or more pathogens are selected from biofilms of Pseudomonas    aeruginosa, Burkholderia cenocepacia, Burkholderia cepacia complex,    Mycobacterium abscessus, Mycobacterium avium, Achromobacter spp.,    Staphylococcus epidermidis, Stenotrophomonas maltophilia, and    Staphylococcus aureus.-   29. The method of embodiment 27, wherein the Pseudomonas aeruginosa    and/or Staphylococcus aureus is multi-drug resistant.-   30. The method of any one of embodiments 9-29, wherein the one or    more pathogens exhibit resistance or is refractory to an antibiotic    selected from amikacin, aztreonam, methicillin, vancomycin,    nafcillin, gentamicin, ampicillin, chloramphenicol, doxycycline,    colistin, delamanid, pretomanid, clofazimine, bedaquiline, and    tobramycin.-   31. The method of embodiment 30, wherein the antibiotic is amikacin    or tobramycin.-   32. The method of embodiment 30, wherein the bacterial pathogen is    methicillin resistant Staphylococcus aureus.-   33. The method of any one of embodiments 1-32, further comprising    co-administering to the subject an antibiotic selected from    amikacin, tobramycin, gentamicin, piperacillin, mezlocillin,    ticarcillin, imipenem, ciprofloxacin, ceftazidime, aztreonam,    ticarcillin-clavulanate, dicloxacillin, amoxicillin,    trimethoprim-sulfamethoxazole, cephalexin, piperacillin-tazobactam,    linezolid, daptomycin, vancomycin, metronidazole, clindamycin,    colistin, tetracycline, levofloxacin, amoxicillin and clavulanic    acid (Augmentin®), cloxacillin, dicloxacillin, cefdinir, cefprozil,    cefaclor, cefuroxime, erythromycin/sulfisoxazole, erythromycin,    clarithromycin, azithromycin, doxycycline, minocycline, tigecycline,    imipenem, meropenem, colistimethate/colistin®, methicillin,    oxacillin, nafcillin, carbenicillin, azlocillin, piperacillin and    tazobactam (Zosyn®), cefepime, ethambutol, rifampin, and meropenem.-   34. The method of embodiment 33, wherein the antibiotic is selected    from meropenem, ceftazidime, tobramycin, amikacin, aztreonam,    ciprofloxacin, colistin, and levofloxacin.-   35. The method of any one of embodiments 9-22, wherein the fungal    pathogen is Candida albicans, drug resistant Candida albicans,    Candida glabrata, Candida krusei, Candida guilliermondii, Candida    auris, Candida tropicalis, Aspergillus niger, Aspergillus terreus,    Aspergillus fumigatus, and/or Aspergillus flavus.-   36. The method of any one of embodiments 1-35, comprising    administering the BT composition via inhalation, orally or nasally,    using an aerosol device.-   37. The method of embodiment 36, wherein the aerosol device is a    nebulizer.-   38. The method of embodiment 36 or 37, wherein the BT composition is    in the form of an aqueous solution, aqueous suspension, or dry    powder.-   39. The method of any one of embodiments 1-38, wherein the BT    composition is administered topically to lung tissue.-   40. The method of any one of embodiments 1-39, wherein

the BT composition is administered as a dosage from about 0.25 mg/mL toabout 15 mg/mL, from about 0.4 mg/mL to about 15 mg/mL, from about 0.6mg/mL to about 15 mg/mL, from about 0.6 mg/mL to about 100 mg/mL, fromabout 5 mg/mL to about 100 mg/mL, from about 10 mg/mL to about 100mg/mL, from about 25 mg/mL to about 100 mg/mL, from about 50 mg/mL toabout 100 mg/mL, from about 0.8 mg/mL to about 15 mg/mL, from about 1mg/mL to about 10 mg/mL, from 2.5 mg/mL to about 10 mg/mL, from about 4mg/mL to about 10 mg/mL, from about 5 mg/mL to about 10 mg/mL, fromabout 6 mg/mL to about 10 mg/mL, 0.6 mg/mL to about 6 mg/mL, from about4 mg/mL to about 15 mg/mL, from about 6 mg/mL to about 15 mg/mL, fromabout 50 μg/mL to about 750 μg/mL, from about 75 μg/mL to about 500μg/mL, from about 100 μg/mL to about 250 μg/mL, from about 100 μg/mL toabout 150 μg/mL, or from about 75 μg/mL to about 150 μg/mL; and/or

the total amount of the BT composition administered to the lungs is fromabout 0.25 mg to about 15 mg, from about 0.4 mg to about 15 mg, fromabout 0.6 mg to about 15 mg, from about 0.8 mg to about 15 mg, fromabout 1 mg to about 10 mg, from 2.5 mg to about 10 mg, from about 4 mgto about 10 mg, from about 5 mg to about 10 mg, from about 6 mg to about10 mg, 0.6 mg to about 6 mg, from about 4 mg to about 15 mg, from about6 mg to about 15 mg, from about 50 μg to about 750 μg, from about 75 μgto about 500 μg, from about 100 μg to about 250 μg, from about 100 μg toabout 150 μg, or from about 75 μg to about 150 μg.

-   41. The method of any one of embodiments 1-40, wherein the BT    composition is administered as a dosage from about 0.6 mg/mL to    about 6 mg/mL.-   42. The method of any one of embodiments 1-41, wherein the BT    composition is administered three times per day, two times per day,    once daily, every other day, once every three days, once every week,    once every other week, once every month, or once every other month.-   43. The method of embodiment 42, wherein the BT composition is    administered once every week.

044. The method of any one of embodiments 1-43, wherein one or more ofthe following cystic fibrosis symptoms is lessened in severity in thesubject: cough, wheezing, breathlessness, bronchiectasis, nasal polyps,hemoptysis, respiratory failure, and pulmonary exacerbation.

-   45. The method of any one of embodiments 1-44, wherein the    bismuth-thiol composition comprises a plurality of microparticles    that comprise a bismuth-thiol (BT) compound, substantially all of    said microparticles having a volumetric mean diameter of from about    0.4 μm to about 5 μm, and wherein the BT compound comprises bismuth    or a bismuth salt and a thiol-containing compound.-   46. The method of embodiment 45, wherein at least 60%, 65%, 70, 75%,    80%, 90%, or 95% of the microparticles have a volumetric mean    diameter of from about 0.4 μm to about 3 μm, or from about 0.5 μm to    about 2 μm, or from about 0.7 μm to about 2 μm, or from about 0.8 μm    to about 1.8 μm, or from about 0.8 μm to about 1.6 μm, or from about    0.9 μm to about 1.4 μm, or from about 1.0 μm to about 2.0 μm, or    from about 1.0 μm to about 1.8 μm.-   47. The method of embodiments 45-46, wherein the bismuth salt is    bismuth nitrate, bismuth subnitrate, or bismuth chloride.-   48. The method of embodiments 45-46, wherein the thiol-containing    compound comprises one or more agents selected from 1,2-ethane    dithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4    dimercaptotoluene, 2,3-butanedithiol, 1 ,3-propanedithiol,    2-hydroxypropanethiol, 1-mercapto-2-propanol, dithioerythritol,    dithiothreitol, cysteamine, and alpha-lipoic acid.-   49. The method of any one of embodiments 45-48, wherein the BT    composition comprises one or more BT compounds selected from BisBAL,    BisEDT, Bis-dimercaprol, BisDTT, Bis-2-mercaptoethanol, Bis-DTE,    Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal,    Bis-Pyr/BDT, BisPyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,    bismuth-1-mercapto-2-propanol, BisEDT/CSTMN (1:1), BisPYR/CSTMN    (1:1), BisBAL/CSTMN (1:1), BisTOL/CSTMN (1:1), and    BisEDT/2-hydroxy-1-propanethiol.-   50. The method of embodiment 49, wherein the BT compound is selected    from one or more of BisEDT, Bis-Bal, Bis-Pyr, Bis-Ery, Bis-Tol,    Bis-BDT, or BisEDT/2-hydroxy-1-propane thiol.-   51. The method of embodiment 50, wherein the BT compound is BisEDT.-   52. The method of embodiment 50, wherein the BT compound is BisBDT    or BisBAL.-   53. The method of any one of embodiments 1-52, wherein the total    amount of the BT composition administered to the deep lung region is    from about 0.25 mg to about 15 mg, from about 0.4 mg to about 15 mg,    from about 0.6 mg to about 15 mg, from about 0.8 mg to about 15 mg,    from about 1 mg to about 10 mg, from 2.5 mg to about 10 mg, from    about 4 mg to about 10 mg, from about 5 mg to about 10 mg, from    about 6 mg to about 10 mg, 0.6 mg to about 6 mg, from about 4 mg to    about 15 mg, from about 6 mg to about 15 mg, from about 50 μg to    about 750 μg, from about 75 μg to about 500 μg, from about 100 μg to    about 250 μg, from about 100 μg to about 150 μg, or from about 75 μg    to about 150 μg.-   54. The method of embodiment 53, wherein the deep lung region is the    deep lung alveoli.-   55. An aerosol comprising a plurality of dispersed liquid droplets    in a gas, said liquid droplets comprising a BT composition    comprising at least one BT compound suspended therein, wherein the    BT compound comprises bismuth and/or a bismuth salt and a    thiol-containing compound; and

wherein at least 60%, 65%, 70, 75%, 80%, 90%, or 95% of the liquiddroplets have a mass median aerodynamic diameter (MMAD) from about offrom about 0.4 μm to about 5 μm.

-   56. The aerosol of embodiment 55, wherein the liquid droplets have a    MMAD of from about 0.4 μm to about 7 μm, or from about 0.5 μm to    about 5 μm, or from about 0.7 μm to about 4 μm, or from about 0.7 μm    to about 3.5 μm, or from about 0.8 μm to about 3.5 μm, or from about    0.9 μm to about 3.5 μm, or from about 0.9 μm to about 3 μm, or from    about 0.8 μm to about 1.8 μm, or from about 0.8 μm to about 1.6 μm,    or from about 0.9 μm to about 1.4 μm, or from about 1.0 μm to about    2.0 μm, or from about 1.0 μm to about 1.8 μm.-   57. The aerosol of embodiment 56, wherein the liquid droplets have a    MMAD of from about 0.8 μm to about 1.6 μm, or from about 0.9 μm to    about 3.5 μm, or from about 0.9 μm to about 3 μm, or from about 0.9    μm to about 1.4 μm, or from about 1.0 μm to about 2.0 μm, or from    about 1.0 μm to about 1.8 μm.-   58. The aerosol of any one of embodiments 55-57, wherein the    plurality of liquid droplets have a D90 of less than about 5 μm.-   59. The aerosol of embodiment 58, wherein the plurality of liquid    droplets have a D90 of less than about 3 μm.-   60. The aerosol of any one of embodiments 55-59, wherein the    plurality of liquid droplets are dispersed in a continuous gas    phase.-   61. The aerosol of any one of embodiments 55-60, wherein the BT    compound comprises bismuth and/or a bismuth salt associated    covalently and/or in a coordination complex with one or more    thiol-containing compounds.-   62. The aerosol of any one of embodiments 55-61, wherein the bismuth    salt is bismuth nitrate, bismuth subnitrate, or bismuth chloride.-   63. The aerosol of any one of embodiments 55-62, wherein the    thiol-containing compound comprises one or more agents selected from    1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione,    dithioerythritol, 3,4 dimercaptotoluene, 2,3-butanedithiol,    1,3-propanedithiol, 2-hydroxypropanethiol, 1-mercapto-2-propanol,    dithioerythritol, dithiothreitol, cysteamine, and alpha-lipoic acid.-   64. The aerosol of any one of embodiments 55-63, wherein the BT    composition comprises one or more BT compounds selected from BisBAL,    BisEDT, Bis-dimercaprol, BisDTT, Bis mercaptoethanol, Bis-DTE,    Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal,    Bis-Pyr/BDT, BisPyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,    bismuth-1-mercapto-2-propanol, BisEDT/CSTMN (1:1), BisPYR/CSTMN    (1:1), BisBAL/CSTMN (1:1), BisTOL/CSTMN (1:1), and    BisEDT/2-hydroxy-1-propanethiol.-   65. The aerosol of embodiment 64, wherein the BT compound is    selected from one or more of BisEDT, Bis-Bal, Bis-Pyr, Bis-Ery,    Bis-Tol, Bis-BDT, or BisEDT/2-hydroxy-1-propane thiol.-   66. The aerosol of embodiment 65, wherein the BT compound is BisEDT.-   67. The aerosol of embodiment 55, wherein the mass median    aerodynamic diameter (MMAD) is determined by laser time of flight    and/or cascade impactor.-   68. The aerosol of any one of embodiments 55-67, wherein the BT    compound is suspended in the liquid droplet.-   69. The aerosol of embodiment 68, wherein the BT compound is BisEDT.-   70. The aerosol of any one of embodiments 55-69, wherein the    droplets further comprise TWEEN 80® (e.g. from about 0.05% to about    1%) and optionally

a buffer (e.g. sodium phosphate or sodium citrate) at a pH of about 7.4;and/or

sodium chloride.

-   71. The aerosol of any one of embodiments 55-70, wherein if    deposited to the deep lung region, the BT compounds have an average    half-life of at least 2 days.-   72. The aerosol of any one of embodiment 71, wherein if deposited to    the deep lung region, the BT compounds have an average half-life of    at least 4 days.-   73. A method of treating, managing or lessening the severity of    cystic fibrosis (CF) symptoms and infections in a subject, the    method comprising administering to the subject a bismuth-thiol (BT)    composition that comprises at least one BT compound, wherein the    composition is a suspension of microparticles having a volumetric    mean diameter (VMD) from about 0.4 μm to about 5 μm and/or a mass    median aerodynamic diameter (MMAD) from about 0.4 μm to about 5 μm.-   74. The method of embodiment 73, wherein the BT compound is BisEDT.-   75. The method of embodiment 74, wherein the BT composition    comprises BisEDT at a concentration greater than about 0.1 mg/mL,    about 0.05% to about 1.0% TWEEN 80®, about 0.05 to 40 mM sodium    chloride, and optionally about 2 to 20 mM sodium phosphate at about    pH. 7.4.-   76. The method of embodiment 75, wherein the BT composition    comprises BisEDT at a concentration greater than about 0.25 mg/mL,    about 0.5% TWEEN 80®, about 10 mM sodium chloride, and about 10 mM    sodium phosphate at about pH 7.4.-   77. The method of any one of embodiments 73-76, wherein when the BT    composition is aerosolized, wherein the aerosolized liquid droplets    have a MMAD from about of from about 0.4 μm to about 5 μm.-   78. The method of embodiment 77, wherein at least 60%, 65%, 70, 75%,    80%, 90%, or 95% of the liquid droplets have a MMAD of from about    0.4 μm to about 7 μm, or from about 0.5 μm to about 5 μm, or from    about 0.7 μm to about 4 μm, or from about 0.7 μm to about 3.5 μm, or    from about 0.8 μm to about 3.5 μm, or from about 0.9 μm to about 3.5    μm, or from about 0.9 μm to about 3 μm, or from about 0.8 μm to    about 1.8 μm, or from about 0.8 μm to about 1.6 μm, or from about    0.9 μm to about 1.4 μm, or from about 1.0 μm to about 2.0 μm, or    from about 1.0 μm to about 1.8 μm.-   79. The method of embodiment 78, wherein the liquid droplets have a    MMAD of from about 0.8 to about 1.6 μm, or from about 0.9 μm to    about 3.5 μm, or from about 0.9 μm to about 3 μm, or from about 0.9    μm to about 1.4 μm, or from about 1.0 μm to about 2.0 μm, or from    about 1.0 μm to about 1.8 μm.-   80. The method of any one of embodiments 77-79, wherein the    plurality of liquid droplets have a D90 of less than about 3 μm.-   81. The method of embodiment 80, wherein the plurality of liquid    droplets have a D90 of less than about 2 μm.-   82. The method of any one of embodiments 77-81, wherein the    plurality of liquid droplets are dispersed in a continuous gas    phase.-   83. The method of any one of embodiments 77-82, wherein the BT    compound comprises bismuth and/or a bismuth salt associated    covalently and/or in a coordination complex with one or more    thiol-containing compounds.-   84. The method of any one of embodiments 77-83, wherein the bismuth    salt is bismuth nitrate, bismuth subnitrate, or bismuth chloride.-   85. The method of any one of embodiments 77-84, wherein the    thiol-containing compound comprises one or more agents selected from    1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione,    dithioerythritol, 3,4 dimercaptotoluene, 2,3-butanedithiol, 1    ,3-propanedithiol, 2-hydroxypropanethiol, 1-mercapto-2-propanol,    dithioerythritol, dithiothreitol, cysteamine, and alpha-lipoic acid.-   86. The method of any one of embodiments 77-85, wherein the BT    composition comprises one or more BT compounds selected from BisBAL,    BisEDT, Bis-dimercaprol, BisDTT, Bis-2-mercaptoethanol, Bis-DTE,    Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal,    Bis-Pyr/BDT, BisPyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,    bismuth-1-mercapto-2-propanol, BisEDT/CSTMN (1:1), BisPYR/CSTMN    (1:1), BisBAL/CSTMN (1:1), BisTOL/CSTMN (1:1), and    BisEDT/2-hydroxy-1-propanethiol.-   87. The method of embodiment 86, wherein the BT compound is selected    from one or more of BisEDT, Bis-Bal, Bis-Pyr, Bis-Ery, Bis-Tol,    Bis-BDT, or BisEDT/2-hydroxy-1-propane thiol.-   88. The method of embodiment 87, wherein the BT compound is BisEDT.-   89. The method of embodiment 87, wherein the BT compound is BisBDT    or BisBAL.-   90. The method of any one of embodiments 77-89, wherein the total    amount of the BT composition administered to the deep lung region is    from about 0.25 mg to about 15 mg, from about 0.4 mg to about 15 mg,    from about 0.6 mg to about 15 mg, from about 0.8 mg to about 15 mg,    from about 1 mg to about 10 mg, from 2.5 mg to about 10 mg, from    about 4 mg to about 10 mg, from about 5 mg to about 10 mg, from    about 6 mg to about 10 mg, 0.6 mg to about 6 mg, from about 4 mg to    about 15 mg, from about 6 mg to about 15 mg, from about 50 μg to    about 750 μg, from about 75 μg to about 500 μg, from about 100 μg to    about 250 μg, from about 100 μg to about 150 μg, or from about 75 μg    to about 150 μg.-   91. The method of embodiment 90, wherein the deep lung region is the    deep lung alveoli.-   92. The method of any one of embodiments 77-91, wherein the total    amount of the BT composition comprising BisEDT administered to the    deep lung alveoli is from about 0.6 mg to about 6 mg,-   93. The method of any one of embodiments 77-92, wherein the    composition is aerosolized via a nebulizer.-   94. The method of embodiment 93, wherein the nebulizer is a jet    nebulizer.-   95. The method of embodiment 94, wherein the jet nebulizer is a PARI    LC PLUS® jet nebulizer or PART LC SPRINT® jet nebulizer.-   96. The method of any one of embodiments 93-95, wherein the    nebulizer has an inlet pressure from about 10 to about 40 psig.-   97. The method of any one of embodiments 93-96, wherein the inlet    flow is from about 3 L/min to about 8 L/min.-   98. The method of any one of embodiments 93-97, wherein the exhaust    air flow is from about 3 L/min to about 8 L/min.-   99. A method of treating, managing or lessening the severity of    symptoms and infections associated with one or more pulmonary    diseases or infections in a subject, the method comprising    administering to the subject a bismuth-thiol (BT) composition that    comprises at least one BT compound.-   100. The method of embodiment 99, wherein the one or more pulmonary    diseases or infections are not the result of or associated with    cystic fibrosis.-   101. The method of embodiment 99 or 100, wherein the subject has at    least one pulmonary infectionand if there are two or more pulmonary    infections, the infections are either concurrent or successive in    order.-   102. The method of embodiment 100 or 101, wherein the pulmonary    infection is in one lung.-   103. The method of any one of embodiments 100-102, wherein the    pulmonary infection is in both lungs.-   104. The method of any one of embodiments 100-103, wherein the    pulmonary infection is in one or more of the three lobes of the    right lung.-   105. The method of any one of embodiments 100-104, wherein the    pulmonary infection is in one or both of the two lobes of the left    lung.-   106. The method of any one of embodiments 100-105, wherein the    pulmonary infection is bronchiectasis infection, pneumonia, valley    fever, allergic bronchopulmonary aspergillosis (ABPA), ventilator    acquired pneumonia, hospital acquired pneumonia, community acquired    pneumonia, ventilator associated tracheobronchitis, lower    respiratory tract infection, non-tuberculous Mycobacteria, anthrax,    legionellosis, pertussis, bronchitis, Bronchiolitis, COPD-associated    infection, and post-lung transplantation.-   107. The method of any one of embodiments 100-106, wherein the    pulmonary infection contains one or more bacterial or fungal    pathogens.-   108. The method of any one of embodiments 101-107, wherein the    pulmonary infection is a CF-related pulmonary infection.-   109. The method of embodiment 108, comprising treating the    CF-related pulmonary infection.-   110. The method of embodiment 108, comprising managing the    CF-related pulmonary infection.-   111. The method of embodiment 108, comprising lessening the severity    of the CF-related pulmonary infection.-   112. The method of any one of embodiments 100-111, wherein the    pulmonary infection is located in or on the lung mucosa, the bronchi    and/or the bronchioles.-   113. The method of any one of embodiments 100-111, wherein the    pulmonary infection is located on the surface of or within a    bacterial biofilm, aggregated bacteria, a fungal biofilm, or    aggregated fungi.-   114. The method of any one of embodiments 100-111, wherein the    pulmonary infection is located in the sputum.-   115. The method of any one of embodiments 107-114, wherein the    bacterial pathogen comprises one or more of gram-positive bacteria    and gram-negative bacteria.-   116. The method of any one of embodiments 107-115, wherein the    bacterial pathogen comprises one or more of a bacterial biofilm and    planktonic bacteria.-   117. The method of embodiment 116, wherein the method comprises at    least one of: (i) reducing the bacterial biofilm, (ii) impairing    growth of the bacterial biofilm, and (iii) preventing reformation of    the bacterial biofilm.-   118. The method of any one of embodiments 107-114, wherein the    fungal pathogen comprises planktonic fungi and/or biofilm fungi.-   119. The method of any one of embodiments 107-118, wherein the BT    composition treats, manages or lessens the severity of the pulmonary    infection by one or both of:

prevention of the infection by the bacterial or fungal pathogen; and

reduction of the bacterial or fungal pathogen; and/or

reducing the viscosity of the sputum.

-   120. The method of any one of embodiments 107-118, wherein the BT    composition treats, manages or lessens the severity of the pulmonary    infection by one or more of:

prevention of elaboration or secretion of exotoxins from the bacterialor fungal pathogen;

inhibition of cell viability or cell growth of planktonic cells of thebacterial or fungal pathogen;

inhibition of biofilm formation by the bacterial or fungal pathogen; and

inhibition of biofilm viability or biofilm growth of biofilm-formingcells of the bacterial or fungal pathogen.

-   121. The method of any one of embodiments 107-120, wherein the one    or more pathogens are selected from Haemophilus influenzae,    Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus    warneri Staphylococcus lugdunensis, Staphylococcus epidermidis,    Streptococcus milleri/anginous, Streptococcus pyogenes,    non-tuberculosis Mycobacterium, Mycobacterium tuberculosis,    Burkholderia spp., Achromobacter xylosoxidans, Pandoraea sputorum,    Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, Haemophilus    pittmaniae, Serratia marcescens, Candidia albacans, Candida    parapsilosis, Candida guilliermondii, Morganella morganii,    Inquilinus limosus, Ralstonia mannitolilytica, Pandoraea apista,    Pandoraea pnomenusa, Pandoraea sputorum, Bdellovibrio bacteriovorus,    Bordetella bronchiseptica, Vampirovibrio chlorellavorus,    Actinobacter baumanni, Cupriadidus metallidurans, Cupriavidus    pauculus, Cupriavidus respiraculi, Delftia acidivordans, Exophilia    dermatitidis, Herbaspirillum frisingense, Herbaspirillum    seropedicae, Klebsiella pneumoniae, Pandoraea norimbergensis,    Pandoraea pulmonicola, Pseudomonas mendocina, Pseudomonas    pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri,    Ralstonia insidiosa, Ralstonia pickettii, Neisseria gonorrhoeae,    NDM-1 positive E. coli, Enterobacter cloaca, Vancomycin-resistant E.    faecium, Vancomycin-resistant E. faecalis, E. faecium, E. faecalis,    Clindamycin-resistant S. agalactiae, S. agalactiae, Bacteroides    fragilis, Clostridium difficile, Streptococcus pneumonia, Moraxella    catarrhalis, Haemophilus haemolyticus, Haemophilus parainfluenzae,    Chlamydophilia pneumoniae, Mycoplasma pneumoniae, Atopobium,    Sphingomonas, Saccharibacteria, Leptotrichia, Capnocytophaga,    Oribacterium, Aquabacterium, Lachnoanaerobaculum, Campylobacter,    Acinetobacter; Agrobacterium; Bordetella; Brevundimonas;    Chryseobacterium; Delftia; Enterobacter; Klebsiella; Pandoraea;    Pseudomonas; Ralstonia, and Prevotella.-   122. The method of embodiment 121, wherein the non-tuberculosis    mycobacterium is selected from Mycobacterium abscessus,    Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium    fortuitum, Mycobacterium gordonae, Mycobacterium kansasii,    Mycobacterium avium complex, Mycobacterium marinum, Mycobacterium    terrae and Mycobacterium cheloni.-   123. The method of embodiment 121, wherein the Burkholderia spp. is    selected from Burkholderia cepacia, Burkholderia multivorans,    Burkholderia cenocepacia, Burkholderia stabilis, Burkholderia    vietnamiensis, Burkholderia dolosa, Burkholderia ambifaria,    Burkholderia anthina, Burkholderia pyrrocinia, Burkholderia    gladioli, Burkholderia ubonensis, Burkholderia arboris, Burkholderia    latens, Burkholderia lata, Burkholderia metallica, Burkholderia    seminalis, Burkholderia contaminans, and Burkholderia diffusa.-   124. The method of embodiment 121, wherein the one or more pathogens    are selected from Pseudomonas aeruginosa, single drug-resistant    Pseudomonas aeruginosa, multi drug-resistant Pseudomonas aeruginosa,    Staphylococcus aureus, single drug-resistant Staphylococcus aureus,    multi drug-resistant Staphylococcus aureus, methicillin resistant    Staphylococcus aureus, Mycobacterium abscessus, Mycobacterium avium,    Haemophilus influenzae, Burkholderia cepacia, Burkholderia    multivorans, Burkholderia cenocepacia, Burkholderia dolosa,    Achromobacter xylosoxidans, Stenotrophomonas maltophilia,    Staphylococcus epidermidis, and Burkholderia vietnamiensis.-   125. The method of embodiment 121, wherein the one or more pathogens    are selected from Haemophilus influenzae, Pseudomonas aeruginosa,    and Staphylococcus aureus.-   126. The method of any one of embodiments 117, 118, or 120, wherein    the one or more pathogens are selected from biofilms of Pseudomonas    aeruginosa, Burkholderia cenocepacia, Burkholderia cepacia complex,    Mycobacterium abscessus, Mycobacterium avium, Achromobacter spp.,    Staphylococcus epidermidis, Stenotrophomonas maltophilia, and    Staphylococcus aureus.-   127. The method of embodiment 125, wherein the Pseudomonas    aeruginosa and/or Staphylococcus aureus is multi-drug resistant.-   128. The method of any one of embodiments 107-127, wherein the one    or more pathogens exhibit resistance or is refractory to an    antibiotic selected from amikacin, aztreonam, methicillin,    vancomycin, nafcillin, gentamicin, ampicillin, chloramphenicol,    doxycycline and tobramycin.-   129. The method of embodiment 128, wherein the antibiotic is    amikacin or tobramycin.-   130. The method of embodiment 128, wherein the bacterial pathogen is    methicillin resistant Staphylococcus aureus.-   131. The method of any one of embodiments 99-130, further comprising    coadministering or conjointly administering to the subject an    antibiotic selected from amikacin, tobramycin, gentamicin,    piperacillin, mezlocillin, ticarcillin, imipenem, ciprofloxacin,    ceftazidime, aztreonam, ticarcillin-clavulanate, dicloxacillin,    amoxicillin, trimethoprim-sulfamethoxazole, cephalexin,    piperacillin-tazobactam, linezolid, daptomycin, vancomycin,    metronidazole, clindamycin, colistin, tetracycline, levofloxacin,    amoxicillin and clavulanic acid (Augmentin®), cloxacillin,    dicloxacillin, cefdinir, cefprozil, cefaclor, cefuroxime,    erythromycin/sulfisoxazole, erythromycin, clarithromycin,    azithromycin, doxycycline, minocycline, tigecycline, imipenem,    meropenem, colistimethate/colistin®, methicillin, oxacillin,    nafcillin, carbenicillin, azlocillin, piperacillin and tazobactam    (Zosyn®), cefepime, ethambutol, rifampin, and meropenem.-   132. The method of embodiment 131, wherein the antibiotic is    selected from meropenem, ceftazidime, tobramycin, amikacin,    aztreonam, ciprofloxacin, colistin, and levofloxacin.-   133. The method of any one of embodiments 107-120, wherein the    fungal pathogen is Candida albicans, drug resistant Candida    albicans, Candida glabrata, Candida krusei, Candida guilliermondii,    Candida auris, Candida tropicalis, Aspergillus niger, Aspergillus    terreus, Aspergillus fumigatus, and/or Aspergillus flavus.-   134. The method of any one of embodiments 99-133, comprising    administering the BT composition by inhalation, orally or nasally,    using an aerosol device.-   135. The method of embodiment 134, wherein the aerosol device is a    nebulizer.-   136. The method of embodiment 134 or 135, wherein the BT composition    is in the form of an aqueous solution or suspension.-   137. The method of any one of embodiments 99-136, wherein the BT    composition is administered topically to lung tissue.-   138. The method of any one of embodiments 99-137, wherein

the BT composition is administered as a dosage from about 0.25 mg/mL toabout 15 mg/mL, from about 0.4 mg/mL to about 15 mg/mL, from about 0.6mg/mL to about 15 mg/mL, from about 0.6 mg/mL to about 100 mg/mL, fromabout 5 mg/mL to about 100 mg/mL, from about 10 mg/mL to about 100mg/mL, from about 25 mg/mL to about 100 mg/mL, from about 50 mg/mL toabout 100 mg/mL, from about 0.8 mg/mL to about 15 mg/mL, from about 1mg/mL to about 10 mg/mL, from 2.5 mg/mL to about 10 mg/mL, from about 4mg/mL to about 10 mg/mL, from about 5 mg/mL to about 10 mg/mL, fromabout 6 mg/mL to about 10 mg/mL, 0.6 mg/mL to about 6 mg/mL, from about4 mg/mL to about 15 mg/mL, from about 6 mg/mL to about 15 mg/mL, fromabout 50 μg/mL to about 750 μg/mL, from about 75 μg/mL to about 500μg/mL, from about 100 μg/mL to about 250 μg/mL, from about 100 μg/mL toabout 150 μg/mL, or from about 75 μg/mL to about 150 μg/mL; and/or

the total amount of the BT composition administered to the lungs is fromabout 0.25 mg to about 15 mg, from about 0.4 mg to about 15 mg, fromabout 0.6 mg to about 15 mg, from about 0.8 mg to about 15 mg, fromabout 1 mg to about 10 mg, from 2.5 mg to about 10 mg, from about 4 mgto about 10 mg, from about 5 mg to about 10 mg, from about 6 mg to about10 mg, 0.6 mg to about 6 mg, from about 4 mg to about 15 mg, from about6 mg to about 15 mg, from about 50 μg to about 750 μg, from about 75 μgto about 500 μg, from about 100 μg to about 250 μg, from about 100 μg toabout 150 μg, or from about 75 μg to about 150 μg.

-   139. The method of any one of embodiments 99-138, wherein the BT    composition is administered as a dosage from about 0.6 mg/mL to    about 6 mg/mL.-   140. The method of any one of embodiments 99-139, wherein the BT    composition is administered three times per day, two times per day,    once daily, every other day, once every three days, once every week,    once every other week, once every month, to once every other month.-   141. The method of embodiment 140, wherein the BT composition is    administered once every week.-   142. The method of any one of embodiments 99-141, wherein one or    more of the following symptoms is lessened in severity in the    subject: cough, wheezing, breathlessness, bronchiectasis, nasal    polyps, hemoptysis, respiratory failure, and pulmonary exacerbation.-   143. The method of any one of embodiments 99-142, wherein the    bismuth-thiol composition comprises a plurality of microparticles    that comprise a bismuth-thiol (BT) compound, substantially all of    said microparticles having a volumetric mean diameter of from about    0.4 μm to about 5 μm, and wherein the BT compound comprises bismuth    or a bismuth salt and a thiol-containing compound.-   144. The method of embodiment 143, wherein at least 60%, 65%, 70,    75%, 80%, 90%, or 95% of the microparticles have a volumetric mean    diameter of from about 0.4 μm to about 3 μm, or from about 0.5 μm to    about 2 μm, or from about 0.7 μm to about 2 μm, or from about 0.8 μm    to about 1.8 μm, or from about 0.8 μm to about 1.6 μm, or from about    0.9 μm to about 1.4 μm, or from about 1.0 μm to about 2.0 μm, or    from about 1.0 μm to about 1.8 μm.-   145. The method of embodiments 142-143, wherein the bismuth salt is    bismuth nitrate, bismuth subnitrate, or bismuth chloride.-   146. The method of embodiments 142-143, wherein the thiol-containing    compound comprises one or more agents selected from 1,2-ethane    dithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4    dimercaptotoluene, 2,3-butanedithiol, 1 ,3-propanedithiol,    2-hydroxypropanethiol, 1-mercapto-2-propanol, dithioerythritol,    dithiothreitol, cysteamine, and alpha-lipoic acid.-   147. The method of any one of embodiments 143-146, wherein the BT    composition comprises one or more BT compounds selected from BisBAL,    BisEDT, Bis-dimercaprol, BisDTT, Bis-2-mercaptoethanol, Bis-DTE,    Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal,    Bis-Pyr/BDT, BisPyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,    bismuth-1-mercapto-2-propanol, BisEDT/CSTMN (1:1), BisPYR/CSTMN    (1:1), BisBAL/CSTMN (1:1), BisTOL/CSTMN (1:1), and    BisEDT/2-hydroxy-1-propanethiol.-   148. The method of embodiment 147, wherein the BT compound is    selected from one or more of BisEDT, Bis-Bal, Bis-Pyr, Bis-Ery,    Bis-Tol, Bis-BDT, or BisEDT/2-hydroxy-1-propane thiol.-   149. The method of embodiment 148, wherein the BT compound is    BisEDT.-   150. The method of embodiment 148, wherein the BT compound is BisBDT    or BisBAL.-   151. The method of any one of embodiments 99-150, wherein the total    amount of the BT composition administered to the deep lung region is    from about 0.25 mg to about 15 mg, from about 0.4 mg to about 15 mg,    from about 0.6 mg to about 15 mg, from about 0.8 mg to about 15 mg,    from about 1 mg to about 10 mg, from 2.5 mg to about 10 mg, from    about 4 mg to about 10 mg, from about 5 mg to about 10 mg, from    about 6 mg to about 10 mg, 0.6 mg to about 6 mg, from about 4 mg to    about 15 mg, from about 6 mg to about 15 mg, from about 50 μg to    about 750 μg, from about 75 μg to about 500 μg, from about 100 μg to    about 250 μg, from about 100 μg to about 150 μg, or from about 75 μg    to about 150 μg.-   152. The method of embodiment 151, wherein the deep lung region is    the deep lung alveoli.-   153. The aerosol of any one of embodiments 55-72, wherein at least    60%, 65%, 70, 75%, 80%, 90%, or 95% of the microparticles have a    volumetric mean diameter of from about 0.6 μm to about 2.5 μm.-   154. The aerosol of embodiment 153, where substantially all of the    microparticles have a VMD of from about 0.6 μm to about 2.5 μm.-   155. The aerosol of any one of embodiments 55-72 or 153-154, wherein    at least 70% of the aerosolized particles have a MMAD of about 0.9    μm to about 3 μm.-   156. The aerosol of any one of embodiments 55-72 or 153-155, wherein    the composition is a suspension of microparticles having a    volumetric mean diameter (VMD) from about 0.6 μm to about 2.5 μm    and/or a mass median aerodynamic diameter (MMAD) from about 0.9 μm    to about 3 μm.-   157. The aerosol of any one of embodiments 55-72 or 153-156, wherein    the bismuth-thiol composition comprises a plurality of    microparticles that comprise a bismuth-thiol (BT) compound,    substantially all of said microparticles having a volumetric mean    diameter of from about 0.4 μm to about 5 μm, and wherein the BT    compound comprises bismuth or a bismuth salt and a thiol-containing    compound.-   158. The aerosol of any one of embodiments 55-72 or 153-157, wherein    after delivering the aerosolized composition to a subject, at least    60%, 65%, 70, 75%, 80%, 90%, or 95% of the BT compound dose is    deposited on the lung.-   159. The aerosol of embodiment 158, wherein at least 80% of the BT    compound dose is deposited on the lung.-   160. The aerosol of embodiment 158, wherein at least 90% of the BT    compound dose is deposited on the lung.-   161. The method of any one of embodiments 1-54 or 73-152, wherein at    least 60%, 65%, 70, 75%, 80%, 90%, or 95% of the microparticles have    a volumetric mean diameter of from about 0.6 μm to about 2.5 μm.-   162. The method of embodiment 161, where substantially all of the    microparticles have a VMD of from about 0.6 μm to about 2.5 μm.-   163. The method of any one of embodiments 1-54, 73-152, or 161-162,    wherein at least 70% of the aerosolized particles have a MMAD of    about 0.9 μm to about 3 μm.-   164. The method of any one of embodiments 1-54, 73-152, or 161-163,    wherein the composition is a suspension of microparticles having a    volumetric mean diameter (VMD) from about 0.6 μm to about 2.5 μm    and/or a mass median aerodynamic diameter (MMAD) from about 0.9 μm    to about 3 μm.-   165. The method of any one of embodiments 1-54, 73-152, or 161-164,    wherein the bismuth-thiol composition comprises a plurality of    microparticles that comprise a bismuth-thiol (BT) compound,    substantially all of said microparticles having a volumetric mean    diameter of from about 0.4 μm to about 5 μm, and wherein the BT    compound comprises bismuth or a bismuth salt and a thiol-containing    compound.-   166. The method of any one of embodiments 1-54, 73-152, or 161-165,    wherein if deposited to the lung (e.g. the deep lung region), the BT    compounds have an average half-life of at least 2 days.-   167. The method of any one of embodiment 166, wherein if deposited    to the deep lung region, the BT compounds have an average half-life    of at least 4 days.-   168. The method of any one of embodiments 1-54, 73-152, or 161-167,    wherein after delivering the aerosolized composition to a subject,    at least 60%, 65%, 70, 75%, 80%, 90%, or 95% of the BT compound dose    is deposited on the lung.-   169. The aerosol of embodiment 168, wherein at least 80% of the BT    compound dose is deposited on the lung.-   170. The aerosol of embodiment 168, wherein at least 90% of the BT    compound dose is deposited on the lung.-   171. A method of treating, managing or lessening the severity of    cystic fibrosis (CF) symptoms and infections in a subject, the    method comprising administering to the subject a bismuth-thiol (BT)    composition that comprises BisEDT suspended therein, wherein    administering the BT composition is via inhalation, orally or    nasally, using an aerosol device.-   172. The method of embodiment 171, wherein the BT composition    comprises a plurality of microparticles wherein at least 70% of said    microparticles having a volumetric mean diameter (VMD) of from about    0.6 μm to about 2.5 μm.-   173. The method of embodiment 172, wherein at least 80% of said    microparticles having a VMD of from about 0.6 μm to about 2.5 μm.-   174.The method of embodiment 172, wherein at least 90% of said    microparticles having a VMD of from about 0.6 μm to about 2.5 μm.-   175. The method of embodiment 172, wherein when the BT composition    is aerosolized, at least 70% of the aerosolized liquid droplets have    a mass median aerodynamic diameter (MMAD) from about of from about    0.9 μm to about 3 μm.-   176. The method of embodiment 173, wherein when the BT composition    is aerosolized, at least 80% of the aerosolized liquid droplets have    a MMAD from about of from about 0.9 μm to about 3 μm.-   177. The method of embodiment 174, wherein when the BT composition    is aerosolized, at least 90% of the aerosolized liquid droplets have    a MMAD from about of from about 0.9 μm to about 3 μm.-   178. The method of embodiment 171, wherein the BT composition    comprises BisEDT at a concentration greater than about 0.1 mg/mL,    about 0.05% to about 1.0% TWEEN 80®, about 0.05 to 40 mM sodium    chloride, and optionally about 2 to 20 mM sodium phosphate at about    pH. 7.4.-   179. The method of embodiment 171, wherein if deposited to the deep    lung region, the BisEDT compounds have an average half-life of about    4 days.-   180. The method of embodiment 171, wherein the subject has at least    one pulmonary infection containing one or more bacterial pathogens    and/or fungal pathogens.-   181. The method of embodiment 180, wherein the method comprises at    least one of: (i) reducing a bacterial biofilm, (ii) impairing    growth of a bacterial biofilm, (iii) preventing initial formation of    the bacterial biofilm, and/or (iv) preventing reformation of the    bacterial biofilm.-   182. The method of embodiment 180, wherein the one or more pathogens    are selected from Haemophilus influenzae, Pseudomonas aeruginosa,    Staphylococcus aureus, Staphylococcus warneri Staphylococcus    lugdunensis, Staphylococcus epidermidis, Streptococcus    milleri/anginous, Streptococcus pyogenes, non-tuberculosis    Mycobacterium, Mycobacterium tuberculosis, Burkholderia spp.,    Achromobacter xylosoxidans, Pandoraea sputorum, Stenotrophomonas    maltophilia, Alcaligenes xylosoxidans, Haemophilus pittmaniae,    Serratia marcescens, Candida albicans, drug resistant Candida    albicans, Candida glabrata, Candida krusei, Candida guilliermondii,    Candida auris, Candida tropicalis, Aspergillus niger, Aspergillus    terreus, Aspergillus fumigatus, Aspergillus flavus, Morganella    morganii, Inquilinus limosus, Ralstonia mannitolilytica, Pandoraea    apista, Pandoraea pnomenusa, Pandoraea sputorum, Bdellovibrio    bacteriovorus, Bordetella bronchiseptica, Vampirovibrio    chlorellavorus, Actinobacter baumanni, Cupriadidus metallidurans,    Cupriavidus pauculus, Cupriavidus respiraculi, Delftia acidivordans,    Exophilia dermatitidis, Herbaspirillum frisingense, Herbaspirillum    seropedicae, Klebsiella pneumoniae, Pandoraea norimbergensis,    Pandoraea pulmonicola, Pseudomonas mendocina, Pseudomonas    pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri,    Ralstonia insidiosa, Ralstonia pickettii, Neisseria gonorrhoeae,    NDM-1 positive E. coli, Enterobacter cloaca, Vancomycin-resistant E.    faecium, Vancomycin-resistant E. faecalis, E. faecium, E. faecalis,    Clindamycin-resistant S. agalactiae, S. agalactiae, Bacteroides    fragilis, Clostridium difficile, Streptococcus pneumonia, Moraxella    catarrhalis, Haemophilus haemolyticus, Haemophilus parainfluenzae,    Chlamydophilia pneumoniae, Mycoplasma pneumoniae, Atopobium,    Sphingomonas, Saccharibacteria, Leptotrichia, Capnocytophaga,    Oribacterium, Aquabacterium, Lachnoanaerobaculum, Campylobacter,    Acinetobacter; Agrobacterium; Bordetella; Brevundimonas;    Chryseobacterium; Delftia; Enterobacter; Klebsiella; Pandoraea;    Pseudomonas; Ralstonia, and Prevotella.-   183. An aerosol comprising a plurality of dispersed liquid droplets    in a gas, said liquid droplets comprising a BT composition    comprising BisEDT compound suspended therein; and

wherein at least 70% of the liquid droplets have a MMAD from about offrom about 0.9 μm to about 3 μm.

-   184. The aerosol of embodiment 183, wherein prior to aerosolization,    the BT composition comprises a plurality of microparticles wherein    at least 70% of said microparticles have a VMD of from about 0.6 μm    to about 2.5 μm.-   185. The aerosol of embodiment 183, wherein least 80% of the liquid    droplets have a MMAD from about of from about 0.9 μm to about 3 μm.-   186. The aerosol of embodiment 183, wherein least 90% of the liquid    droplets have a MMAD from about of from about 0.9 μm to about 3 μm.-   187. The aerosol of embodiment 185, wherein prior to aerosolization,    the BT composition comprises a plurality of microparticles wherein    at least 80% of said microparticles have a VMD of from about 0.6 μm    to about 2.5 μm.-   188. The aerosol of embodiment 186, wherein prior to aerosolization,    the BT composition comprises a plurality of microparticles wherein    at least 90% of said microparticles have a VMD of from about 0.6 μm    to about 2.5 μm.-   189. The aerosol of embodiment 183, wherein the droplets further    comprise TWEEN 80® (e.g. from about 0.05% to about 1%) and    optionally a buffer (e.g. sodium phosphate or sodium citrate) at a    pH of about 7.4; and/or sodium chloride.-   190. The aerosol of embodiment 183, wherein if deposited to the deep    lung region, the BisEDT compounds have an average half-life of more    than 2 days.-   191. The aerosol of embodiment 183, wherein if deposited to the deep    lung region, the BisEDT compounds have an average half-life of about    4 days.-   193. A pharmaceutical composition comprising bismuth-thiol (BT)    composition that comprises BisEDT suspended therein, wherein the BT    composition comprises a plurality of microparticles, wherein the D90    of said microparticles is less than or equal to 1.9 μm.-   194. A pharmaceutical composition comprising bismuth-thiol (BT)    composition comprises BisEDT suspended therein, wherein the BT    composition comprises a plurality of microparticles, wherein the D90    of said microparticles is less than or equal to about 1.6 μm.-   195. The pharmaceutical composition of embodiment 193, wherein at    least 70% of said microparticles having a volumetric mean diameter    of from about 0.6 μm to about 2.5 μm.-   196. The pharmaceutical composition of embodiment 193, wherein at    least 90% of said microparticles having a volumetric mean diameter    of from about 0.6 μm to about 2.5 μm.-   197. A method of treating, managing or lessening the severity of    symptoms and infections associated with one or more pulmonary    diseases or infections in a subject, the method comprising    administering to the subject a bismuth-thiol (BT) composition that    comprises BisEDT, wherein the BT composition comprises a plurality    of microparticles wherein at least 70% of said microparticles having    a volumetric mean diameter of from about 0.6 μm to about 2.5 μm, and    wherein when the BT composition is aerosolized, at least 70% of the    aerosolized liquid droplets have a MMAD from about of from about 0.9    μm to about 3 μm.-   198. The method of embodiment 197, wherein the one or more pulmonary    diseases or infections are not the result of or associated with    cystic fibrosis.-   199. The method of embodiment 198, wherein the pulmonary infection    is bronchiectasis infection, pneumonia, valley fever, allergic    bronchopulmonary aspergillosis (ABPA), ventilator acquired    pneumonia, hospital acquired pneumonia, community acquired    pneumonia, ventilator associated tracheobronchitis, lower    respiratory tract infection, non-tuberculous Mycobacteria, anthrax,    legionellosis, pertussis, bronchitis, Bronchiolitis, COPD-associated    infection, and post-lung transplantation.

EXAMPLES

The following examples are provided to illustrate the presentdisclosure, and should not be construed as limiting thereof. Additionalexperimental procedures and details can be found in International PatentApplication Nos. PCT/US2010/023108, PCT/US2011/023549, andPCT/US2011/047490, which are hereby incorporated by reference in theirentireties for all purposes.

Example 1 General Synthesis of BisEDT

The starting materials and reagents used in preparing these compoundsare either available from commercial supplier such as Aldrich ChemicalCo., Bachem, etc., or can be made by methods well known in the art. Thestarting materials and the intermediates and the final products of thereaction can be isolated and purified if desired using conventionaltechniques, including but not limited to filtration, distillation,crystallization, chromatography, and the like and can be characterizedusing conventional means, including physical constants and spectraldata. Unless specified otherwise, the reactions described herein takeplace at atmospheric pressure over a temperature range from about −78°C. to about 150° C.

Microparticulate bismuth-1,2-ethanedithiol (BisEDT, soluble bismuthpreparation) was prepared as follows: To an excess (11.4 L) of 5%aqueous HNO₃ at room temperature in a 15 L polypropylene carboy wasslowly added by dropwise addition 0.331 L (˜0.575 moles) of an aqueousBi(NO₃)₃ solution (43% Bi(NO₃)₃ (w/w), 5% nitric acid (w/w), 52% water(w/w), Shepherd Chemical Co., Cincinnati, Ohio, product no. 2362; δ˜1.6g/mL) with stirring, followed by slow addition of absolute ethanol (4L). Some white precipitate formed but was dissolved by continuedstirring. An ethanolic solution (˜1.56 L, ˜0.55 M) of 1,2-ethanedithiol(CAS 540-63-6) was separately prepared by adding, to 1.5 L of absoluteethanol, 72.19 mL (0.863 moles) of 1,2-ethanedithiol using a 60 mLsyringe, and then stirring for five minutes. The 1,2-ethanedithiol/EtOHreagent was then slowly added by dropwise addition over the course offive hours to the aqueous Bi(NO₃)₃/HNO₃ solution, with continuedstirring overnight. The formed product was allowed to settle as aprecipitate for approximately 15 minutes, after which the filtrate wasremoved at 300 mL/min using a peristaltic pump. The product was thencollected by filtration on fine filter paper in a 15-cm diameter Buchnerfunnel, and washed sequentially with three, 500-mL volumes each ofethanol, USP water, and acetone to obtain BisEDT (694.51 gm/mole) as ayellow amorphous powdered solid. The product was placed in a 500 mLamber glass bottle and dried over CaCl₂ under high vacuum for 48 hours.Recovered material (yield ˜200 g) gave off a thiol-characteristic odor.The crude product was redissolved in 750 mL of absolute ethanol, stirredfor 30 min, then filtered and washed sequentially with 3×50 mL ethanol,2×50 mL acetone, and washed again with 500 mL of acetone. The rewashedpowder was triturated in 1M NaOH (500 mL), filtered and washed with3×220 mL water, 2×50 mL ethanol, and 1×400 mL acetone to afford 156.74gm of purified BisEDT. Subsequent batches prepared in essentially thesame manner resulted in yields of about 78-91%.

The product was characterized as having the structure shown above byanalysis of data from ¹H and ¹³C nuclear magnetic resonance (NMR),infrared spectroscopy (IR), ultraviolet spectroscopy (UV), massspectrometry (MS) and elemental analysis. An HPLC method was developedto determine chemical purity of BisEDT whereby the sample was preparedin DMSO (0.5 mg/mL). The λ_(max) was determined by scanning a solutionof BisEDT in DMSO between 190 and 600 nm. Isocratic HPLC elution at 1mL/min was performed at ambient temperature in a mobile phase of 0.1%formic acid in acetonitrile:water (9:1) on a Waters (Millipore Corp.,Milford, Mass.) model 2695 chromatograph with UV detector monitoring at265 nm (λ_(max)), 2 μL injection volume, equipped with a YMC Pack PVCSil NP, 5 μm, 250×4.6 mm inner diameter analytical column (Waters) and asingle peak was detected, reflecting chemical purity of 100±0.1%.Elemental analysis was consistent with the structure of BisEDT as shownabove.

The dried particulate matter was characterized to assess the particlesize properties. Briefly, microparticles were resuspended in 2%Pluronic® F-68 (BASF, Mt. Olive, N.J.) and the suspension was sonicatedfor 10 minutes in a water bath sonicator at standard setting prior toanalysis using a Nanosizer/Zetasizer Nano-S particle analyzer (modelZEN1600 (without zeta-potential measuring capacity), MalvernInstruments, Worcestershire, UK) according to the manufacturer'srecommendations. From compiled data of two measurements, microparticlesexhibited a unimodal distribution with all detectable events betweenabout 0.6 microns and 4 microns in volumetric mean diameter (VMD) andhaving a peak VMD at about 1.3 microns.

Example 2 Preparation of Microparticulate bismuth-1-2-ethanedithiol(BisEDT)

Microparticulate bismuth-1,2-ethanedithiol (BisEDT) was prepared asfollows: Water (25.5 L) and 70% nitric acid (1800 mL) were mixedtogether in a Nalgene reactor. Then, water (2300 mL) was added to anErlenmeyer flask, followed by bismuth subnitrate (532 g), and themixture was stirred. To the mixture was added 70% nitric acid (750 mL)to obtain a clear solution. This solution was transferred into theNalgene reactor and the resulting mixture was stirred for 20 min. Then,9.5 L of 95% EtOH was added to the reactor in three portions.

Separately, 1,2-ethanedithiol, 98%, (229 mL) was added to a bottlefollowed by two 250 mL EtOH portions with stirring. A further 5 L EtOHwas added to the bottle with stirring. The 1,2-ethanedithiol solutionwas then added to the reactor over about 4 hours while stirring. Afterstirring for 18 hours, the solids were allowed to settle for 2 hours.EtOH (20 L) was added and the mixture stirred for 24 hours. The solidswere allowed to settle for 1.5 hours, then separated by filtration ofthe mixture, followed by rinsing with EtOH.

To the empty reactor was added 9 L EtOH and the filtered solids, whichwas stirred for 18 hours. The solids were allowed to settle for 1 hour,then separated by filtration of the mixture, followed by rinsing withEtOH. Next, the empty reactor was charged with 9 L acetone, 99.5%, andthe filtered solids, which was stirred for 15 hours. The solids wereallowed to settle for 1.5 hours, then separated by filtration of themixture, followed by rinsing with acetone. Again, the empty reactor wascharged with 9 L acetone, 99.5%, and the filtered solids, which wasstirred for 1.4 hours. The solids were filtered and air-dried for 69hours, then vacuum-dried for 4 hours. After mixing the solid, it wassieved through a 10 mesh (2 mm) and then 18 mesh (1 mm) sieve to giveBisEDT.

Example 3 Synthesis of Additional Bismuth Thiol Compounds

The following bismuth thiol compounds can also be prepared according tothe methods of Examples 1 and 2:

bismuth-2,3-dimercaptopropanol (2:3 molar ratio, BisBAL)bismuth-4-methyl-1,2-benzenedithiol (2:3 molar ratio, BisTOL)bismuth-2,3-butanedithiol (BisBDT)

Example 4 Formulation(s) for CF Inhalant

Objective: The objective of this study was to develop methods for thenose-only inhalation exposures of BisEDT for rodents. The aerosols weregenerated from suspension formulations of BisEDT in aqueous media (0.5%TWEEN 80® in Sodium Phosphate Buffer, pH 7.4). Aerosols were generatedwith a commercial compressed air jet nebulizer into a rodent nose onlyinhalation exposure system. Suspension formulations were created bysonication with neat API with 0.5% TWEEN 80®in Sodium Phosphate Buffer,pH 7.4. This was further refined by adjusting the vehicle to anosmolality of 300 mOsmol/kg. Aerosols were characterized for particlesize distribution and aerosol concentration. Aerosol concentration wasdetermined by differential mass and chemical analysis of the aerosolfilter samples.

Suspension concentrations between 2.5 and 100 mg/mL were evaluated forone formulation of BisEDT and ranged from 18.5 to 719 μg/L respectively.Further tests were performed with a new formulation of BisEDT and thesuspension concentrations of 2.5 mg/mL, 10 mg/mL, 50 mg/mL and 100 mg/mLyielded aerosol concentrations of 18.4, 97.7, 159, and 1300 μg/L,respectively. The particle size increased with suspension concentrationfrom ˜1 μm to 3.5 μm MMAD, which is respirable for rodent inhalationexposures.

To support potential dose range finding studies in rat nose onlyinhalation exposures are typically recommended for between 30 and 180minutes. Therefore the pulmonary deposited dose range from thesesuspension formulations are between 42 μg/kg and 17.5 mg/kg. Asappropriate the suspension concentration and/or exposure time can bemodulated to adjust pulmonary deposited dose.

Materials and Methods: BisEDT—Two separate lots of Bismuth EthaneDithiol, BisEDT, were used as received. The initial stages of methoddevelopment were performed with Lot #ED268-1-11-01 and the final stageswere performed with Lot #XL-47-125, which had been shipped inpreparation for exposures. The two materials behaved slightlydifferently, requiring a change in the final determination of exposuretimes.

Aerosol Methods: Formulation—Suspension formulations of BisEDT wereprepared in 0.5% TWEEN 80® in Sodium Phosphate Buffer, pH 7.4.Suspensions of 2.5, 10, 25, 50, 75 and 100 mg/mL were prepared bysonication with a Covaris sonicator. In later tests, the vehicle wasadjusted with sodium chloride to achieve an Osmolality of 300 mOsmol/kg.Suspensions were prepared at 2.5, 50, and 100 mg/mL.

Aerosol Delivery System: FIG. 1 shows a schematic of the aerosolgeneration and exposure system used during these tests. Aerosol wasgenerated by using a PARI LC PLUS® compressed air jet nebulizer (PariRespiratory Equipment Inc., Midlothian, Va.) with an inlet pressure of20 psi. The aerosols transitioned into a rodent nose-only inhalationexposure system operated with an inlet air flow of ˜8.5 L/min and anexhaust air flow of ˜7.1 L/min. Aerosol generated by the nebulizer wasdelivered to a one-tier nose-only flow-past inhalation exposure system.

Aerosol concentration was measured at the breathing zone of the exposuresystem by collection of the aerosol onto 47-mm filters (Whatman GF/Amembrane filters). Filter samples were collected at a nominal flowrateof 0.3 L/min. Aerosol mass concentration was determined by differentialweight analysis of the filter samples. Filters were then transferred tothe analytical chemistry laboratory for analysis using HPLC. Details ofthe analytical chemistry methods are included in analytical methodACM-1046-0 (Determination of BisEDT in Formulations and Filter Extractsby HPLC-UV).

Particle Size Distribution Measurement: Particle size distribution forexposure was measured by using an In-Tox Mercer 2.0 L/min cascadeimpactor (In-Tox, LLC, Moriarty, N. Mex.).

Calculation of Deposited Dose: The first two equations were used tocalculate the presented aerosol dose and the theoretical deposited dose,respectively. In these calculations the average aerosol concentration(chemistry) along with projected body weights for rats are used.

${D_{P}\left\lbrack {{mg}{kg}^{- 1}} \right\rbrack} = \frac{{{AC}\left\lbrack {{mg}L^{- 1}} \right\rbrack}{{RMV}\left\lbrack {L\min^{- 1}} \right\rbrack}{T\left\lbrack \min \right\rbrack}}{{BW}\lbrack{kg}\rbrack}$${D_{D}\left\lbrack {{mg}{kg}^{- 1}} \right\rbrack} = \frac{{{AC}\left\lbrack {{mg}L^{- 1}} \right\rbrack}{{RMV}\left\lbrack {L\min^{- 1}} \right\rbrack}{T\left\lbrack \min \right\rbrack}{DF}}{{BW}\lbrack{kg}\rbrack}$RMV = 0.608BW^(0.852)

Where: D_(P): Presented dose; D_(D): Deposited Dose; AC: AerosolExposure Concentration; RMV: Respiratory Minute Volume (Alexander. D J,et al., 2008. Inhal Toxicol.; 20(13): 1179-89); T: Exposure time; BW:Body Weight; DF: Pulmonary deposition fraction (assumed 10%, Tepper etal., 2016 Int J Toxicol; 35(4):376-392); Time varied between 30 and 180minutes.

Results

Aerosol Concentration and Particle Size: The average total aerosolconcentration, BisEDT aerosol concentration and particle size for theformulations are shown for the first BisEDT formulation (Lot#ED268-1-11-01) in Table 1. An analogous table is shown for the secondBisEDT formulation (Lot #XL-47-125) in Table 2. An example histogram ofthe particle size distribution for the 2.5 mg/mL concentration taken viacascade impactor is shown in FIG. 2 . Example histograms of the particlesize distributions for the 25, 50, 75, and 100 mg/mL concentrationstaken via APS are shown in FIGS. 3, 4, 5, and 6 ; respectively. Repeatedtests for size distributions for the second BisEDT formulation aredepicted in FIGS. 7, 8, 9, and 10 for 100 mg/mL, 50 mg/mL, 10 mg/mL, and2.5 mg/mL respectively.

TABLE 1 Summary of suspension aerosol testing for BisEDT (Lot #ED268-1-11-01) Formulation Total Aerosol BisEDT Aerosol Conc. Particle(mg/mL) Conc. (mg/L) (μg/L) Size (GSD)*  1 9.9 1.65 (1.6) μm   2.5 0.11618.5 0.97 (3.0) μm 10 0.184 80.7 N/A 25 0.441 87.9 (64.4)⁺ 2.98 (1.8) μm50 0.599 156 2.83 (1.9) μm 75 0.618 530 3.16 (1.70) μm  100  0.816 7193.53 (1.71) μm  100* 0.731 568 3.60 (1.71) μm  *All Particle size datacollected with an APS except the 2.5 mg/mL formulation ⁺for one of thefilters of the 25 mg/mL solution, chemical extraction yielded a lessthan expected concentration of BisEDT. 64.4 μg/L is the average value ofall extracted concentrations, while 87.9 μg/L is the average excludingthis outlier.

TABLE 2 Summary of suspension aerosol testing for BisEDT (Lot #XL-47-125) using a 300 mOsmol/kg vehicle Formulation Total AerosolBisEDT Aerosol Conc. Particle (mg/mL) Conc. (mg/L) (μg/L) Size (GSD) 1001.535 1300 2.90 (1.67) μm 100 1.404 N/A 2.60 (1.65) μm 50 0.711 159 2.79(1.69) μm 10 0.332 97.7 1.61 (1.65) μm 2.5 0.175 18.4 1.44 (1.71) μm

The aerosols in Tables 1 and 2 were generated with a PARI LC PLUSPERFORMANCE® nebulizer. The nebulizer was tested with a 1.2 barcompressor; measured with Malvern MasterSizer X at 50% relativehumidity, 0.9 NaCl solution, inspiratory flow of 20 L/min, continuousnebulization at 23° C. with a fill volume of 2.5 mL.

Pulmonary Deposited Dose: Pulmonary deposited doses can be modulated viathe aerosol concentration and/or the exposure duration. For a rodentnose-only inhalation exposure, the exposure duration is typicallytargeted between 30 and 180 minutes. Using the performance of theaerosol and the pulmonary dose equation above, the pulmonary depositeddose range for the first BisEDT formulation would be between 42 μg/kgand 9.6 mg/kg. The second formulation was more efficient in depositingBisEDT and is capable of giving a dose up to 17.5 mg/kg.

Methods for formulation and aerosol generation of BisEDT were developed.They support a pulmonary deposited dose between 42 μg/kg and 17.5 mg/kg.These exposure conditions are appropriate for rodent inhalationexposures.

Example 5 In Vitro Studies

This Example describes a series of experiments evaluating thesuitability and feasibility of the development of bismuth-thiols,particularly BisEDT, as an inhaled drug product for the treatment ofCF-related pulmonary infections. A CF-relevant range of infectiousbacterial pathogens, including highly resistant strains and biofilmforming strains of such pathogens, were tested for susceptibility tobismuth-thiols. In addition, in vitro evaluations of toxicity againstlung airway epithelial cells were conducted.

Standardized microbiological susceptibility testing was carried out withtwo bismuth-thiol compounds against a range of the mostclinically-challenging, highly resistant CF isolates. MIC testing wasstandardized using ATCC strains of E. coli (25922) and S. aureus(29213).

The results of this testing demonstrated BisEDT was consistently potentagainst all CF isolates tested, including aminoglycoside-resistant andMDR P. aeruginosa, B. cenocepacia, Achromobacter spp., and S.maltophilia, with all test organisms demonstrating MICs of less than 1μg/mL (see FIG. 12 ).

To expand upon this, the tested bacteria was expanded to include moreBurkholderia spp., as well as to test a wider range of CF isolates andantibiotic-resistant strains.

A range of CF-isolated strains of Mycobacterium avium and Mycobacteriumabscessus were included; which allows for understanding of the activityagainst non-tubercular Mycobacteria (NTM). The results of this study areshown below. Two distinct bismuth-thiol compounds were evaluated,including BisEDT and BisBDT. Mycobacterium avium isolates were found tohave the highest MICs, though BisEDT demonstrated lower MICs againstthese NTM bacteria than did BisBDT. The CF-isolates of greater clinicalrelevance, including both M. abscessus spp. and Burkholderia spp., wereboth consistently found to be much more susceptible to BisEDT andBisBDT. This study demonstrated very compelling MIC data againstCF-isolates of both NTM and Burkholderia spp., as few commercialantibiotics have this level and spectrum of activity.

TABLE 3A BisEDT and BisBDT MIC against bacterial strains BisEDT BisBDTStrain MIC MIC Designation Species Strain characteristics (μg/mL)(μg/mL) MAC101 M. avium MAC reference strain, blood isolate from HIV 2 1patient AMT 0193-13 M. avium MAC CF isolate, macrolide-S 8 >16 AMT0119-8 M. avium MAC CF isolate, macrolide-S 8 >16 BC 5 B. multivorans(cepacia complex) CF isolate, beta lactam-S, TMP/SMX-S, FQ-I 1 2 PC 213B. cepcacia complex (not further CF isolate, beta lactam-S, TMP/SMX-S,FQ-S 0.25 1 speciated) BC 9 B. cepcacia complex (not further CF isolate,beta lactam-S, TMP/SMX-S, FQ-R 0.5 0.5 speciated) BC 17 B. cepacia(cepacia complex) CF isolate, beta lactam-R, TMP/SMX-R, FQ-R 8 4 BC 11B. dolosa (cepacia complex) CF isolate, beta lactam-R, TMP/SMX-S, FQ-R0.125 0.5 ATCC 19977 M. abscessus abscessus ATCC type strain 0.5 0.25AMT 0136-10 M. abscessus complex MABSC CF isolate, macrolide-R,amikacin-R 0.125 0.0625 AMT 0089-5 M. abscessus complex MABSC CFisolate, macrolide-R, amikacin-R 0.25 0.125 AMT 0166-29 M. abscessuscomplex MABSC CF isolate, macrolide-R, amikacin-R 0.125 0.125 AMT0157-14 M. abscessus complex MABSC CF isolate, macrolide-R, amikacin-I0.125 0.125 AMT 0130-8 M. abscessus complex MABSC CF isolate,macrolide-S, amikacin-I 0.125 0.0625 AMT 0153-9 M. abscessus complexMABSC CF isolate, macrolide-S, amikacin-I 0.25 0.125 AMT 0068-40 M.abscessus complex MABSC CF isolate, macrolide-S, amikacin-I 0.25 0.125AMT 0119-7 M. abscessus complex MABSC CF isolate, macrolide-S,amikacin-I 0.125 0.125 AMT 0493-2 M. abscessus complex MABSC CF isolate,macrolide-S, amikacin-R 0.5 0.5

TABLE 3B BisEDT and BisBDT MIC against control bacterial strainsExpected results: goal (acceptable range) mcg/mL (see detailed data formeasured ATCC strains used to MICs with each assay set-up) control fordrug activity BisEDT MIC BisBDT MIC ATCC 27853 P. aeruginosa 1-2 (0.5-4)NA ATCC 25922 E. coli   1 (0.5-2) NA ATCC 29213 S. aureus 0.25-0.5 0.25(0.125-1) (0.125- 

indicates data missing or illegible when filed

Controlled in vitro evaluation of BisEDT, with respect to potentialcytotoxicity on fully differentiated human airway epithelium was carriedout. Both solubilized and solid (powder/particulate) forms of BisEDTwere evaluated for cytotoxicity by Epithelix, a contract researchorganization (CRO) specializing in this form of cytotoxicity evaluationutilizing a proprietary, in vitro fully differentiated human airwayepithelium test system (MUCILAIR™). The MUCILAIR™ system facilitatedevaluation of cytotoxicity through both LDH release (from the culturemedium side) and trans-epithelial electrical resistance (TEER) from theapical/air-exposed side as well as pre- and post-exposure microscopicexamination for morphological changes (FIG. 13 ).

With respect to evaluation of six concentrations of solubilized BisEDT,no changes in cellular morphology were noted at four different timepoints (up to 48 hours), nor were changes noted in LDH release and TEER,indicating that no toxic effects were observed, at all time points evenat the highest tested concentration of 30 uM (20.83 ug/mL, which isapproximately 20-347 fold higher than the MICs recently derived, againstmost tested CF bacterial isolates, including M. abscessus (includingamikacin- and clarithromycin/macrolide-resistant strains), Burkholderiaspp. (including beta-lactam, fluoroquinolone-, andsulfamethoxazole-resistant strains), P. aeruginosa (includingmultidrug-resistant (MDR) strains), Achromobacter spp., Stenotrophomonasmaltophilia, and S. aureus (including MRSA)). The highest testedconcentration of 20.83 μg/mL is also approximately 2.5-10 fold higherthan the MIC forts. avium, which comparatively, had the highest MICsrelative to BisEDT of any tested CF isolates. Additionally, since nohint of toxicity has been demonstrated with even the highestconcentration of solubilized BisEDT, it is possible that higherconcentrations may also be determined to benon-toxic/safe/well-tolerated.

With respect to evaluation of five concentrations of particulate BisEDTdiluted in dextran as a carrier (weight/area), the lower twoconcentrations did not produce changes in cellular morphology at thefour different time points (up to 48 hours), nor were changes noted inLDH release and TEER, indicating that no toxic effects were observed atany of the four time points.

With respect to morphology, the top three concentrations demonstratedchanges that were more pronounced in the top two concentrations. Withrespect to the top three concentrations, no significant effect was notedwith respect to TEER at any of these concentrations at the 1 hour timepoint, but at 8, 24, and 48 hours, TEER, loss of tissue integrityoccurred with all three of the highest concentrations. Similarly, noeffect on LDH release was noted at any time point with the lower twoconcentrations, nor at 1 hour for the top three concentrations; butbeyond the 1 hour time point, there was a time and dose dependentincrease in LDH release and decrease in TEER, indicating cytotoxicityunder these conditions. It is notable that in Phase 1 human clinicalstudies, concentration of up to 2500 μg/cm² (˜8-fold higher than the 333μg/cm², the highest concentration tested in this in vitro study) waswell-tolerated when administered topically humans for 6 hours, and aconcentration of up to 75 μg/cm² (intermediate between 33.3 μg/cm² and333 μg/cm²) was tolerated when administered topically to humans for over21 days continuous exposure to normal and abraded skin. This indicatesthat in vitro model conditions may be much more sensitive to theparticulate form than in vivo physiologic conditions, thusover-representing the true risk of cytotoxicity of the particulate form.Nevertheless, the highest non-toxic concentrations of both forms ofBisEDT tested in this in vitro cytotoxicity model, whether particulateor solubilized, provides 25-345 fold the MIC needed to be effectiveagainst a broad spectrum of antibiotic-resistant, difficult to treat CFbacterial pathogens.

The data from this study demonstrates that the solubilized version ofBisEDT is safe at sufficient multiples of the anticipated clinical dose.

Activity of BT Compounds Against CF-Isolate Biofilms

The activity of BT compounds against biofilms grown from CF-isolates wastested. MR14 is a multidrug-resistant (MDR) CF-isolate of Pseudomonasaeruginosa. Reductions in biofilm cell viability of 2 logs (MB-6) to 4logs (MB-1-B3) occurred at 25 ng/mL (FIG. 14 ). The bismuth-thiolcompounds have previously been reported in the literature to haveanti-biofilm effects at sub-inhibitory concentrations with 24 hourtreatment with 0.25 μg/mL. There is nothing comparable in the scientificliterature.

AG14 is an aminoglycoside-resistant CF-isolate of Pseudomonasaeruginosa. Reductions in biofilm cell viability of 2 logs (MB-6) toapproximately 3.5 logs (MB-1-B3) occurred at 0.25 μg/mL. Once again, avery advanced level of anti-biofilm activity (a 6 log reduction) with 24hour treatment occurred with 0.25 μg/mL; this potent level of activityis very likely to be unique to the bismuth-thiol compounds (FIG. 15 ).

Combined, the results of testing against Pseudomonal biofilms (MR14 andAG14) demonstrate an advanced, possibly unique level of anti-biofilmactivity against antibiotic- and multidrug-resistant (MDR) Pseudomonasaeruginosa; this may represent an important new therapeutic activity andclinical strategy in the treatment of pulmonary infections associatedwith cystic fibrosis.

AU197 is a CF-isolate of B. cenocepacia. While in this case, theanti-biofilm activity is not occurring at a subinhibitory level (as withthe previous examples of P. aeruginosa), this level of anti-biofilmactivity (a 6 log reduction at a concentration of 2.5 μg/mL of BisEDT)is nevertheless extremely potent, and is very likely to betherapeutically achievable (FIG. 16 ).

AMT0130-8 represents a CF-isolate of the clinically relevant MABSC,which frequently complicates the treatment of CF pulmonary infections.In this case, while BisBDT demonstrated only very modest reductions inbiofilm cell viability, once again, BisEDT demonstrated a 6 logreduction at 2.5 μg/mL, as well as a dose response from 2.5 ng/mL to 2.5μg/mL. Interestingly, the MIC against this strain was demonstrated to belower for BisBDT (0.0625 μg/mL) than for BisEDT (0.125 μg/mL), yet theanti-biofilm activity of BisEDT was apparently demonstrated to be muchmore potent—while it is not surprising to see such differences inactivity between distinct bismuth-thiol compounds, this particular MABSCstrain was apparently technically difficult to work with (see bulletpoint notes below figure), which may also have accounted for such(apparent) differences in activity (FIG. 17 ).

AMT0089-5 is a macrolide-resistant, amikacin-resistant MABSC. Theinvolvement of such antibiotic-resistant strains of MAB SC in thepulmonary infections of CF patients is extremely problematic. Here,while BisEDT showed a 2.5 log reduction in viable biofilm cells at aconcentration of 2.5 μg/mL, BisBDT was demonstrated to have reduceviable biofilm cells by 6 logs at a concentration of 2.5 μg/mL (FIG. 18).

ATCC-19977 is M. abscessus (macrolide-resistant; inducible). A doseresponse is demonstrated showing a 3 log reduction in viable biofilmcells at 2.5 μg/mL ATCC-19977 is M abscessus (macrolide-resistant;inducible). A dose response is demonstrated below, with a 3 logreduction in viable biofilm cells at 2.5 μg/mL (FIG. 19 ).

The bismuth-thiols were not observed to be active against biofilm formedby a MABSC CF isolate, though this strain was so slow-growing, a longerexposure to the bismuth-thiol compounds may have been necessary todemonstrate activity (FIG. 20 ).

Achromobacter spp. were tested up to concentrations of 250 μg/mL of bothBisEDT and BisBDT, which resulted in a 5 log reduction in viable biofilmcells. A dose response is also apparently associated with both compounds(FIG. 21 ).

Unfortunately, no activity was apparent for either bismuth-thiolcompound against Stenotrophomonas maltophilia (FIG. 22 ).

Finally, anti-biofilm activity was also demonstrated at the highestconcentrations of both compounds (a 5 log reduction) when tested againstE. coli (FIG. 23 ).

Both BisEDT and BisBDT are demonstrated to have very low MIC valuesagainst M. abscessus, MDR P. aeruginosa, Achromobacter spp., andBurkholderia spp. As before, ATCC control strains are utilized tostandardize the data. However, in this evaluation, the bismuth-thiolswere compared head to head with amikacin and clarithromycin (aclinically important macrolide antibiotic). As can be seen from thisdata below in Table 4, the bismuth-thiols are notably and consistentlymore potent than both amikacin and clarithromycin (most dramaticallywhen considering the MABSC strain ATCC 19977, which was induced to bemacrolide-resistant).

TABLE 4A Comparison of conventional antibiotics vs BisEDT and BisBDTactivity against bacterial strains SUMMARY OF MIC RESULTS Special StrainMIC MIC MIC MIC Strain characteristics (mcg/mL) (mcg/mL) (mcg/mL)(mcg/mL) Designation Species (if any) Amikacin Clarithromycin Bis-EDTBis-BDT ATCC 19977 M abscessus/massiliense Macrolide resistant   8 ² >32²  0.06 ²   <0.03 ² complex (inducible) AMT0130-8 Mabscessus/massiliense  16 ² 1 0.06 ²   <0.03 ² complex AMT153-9 Mabscessus/massiliense  32 ² 2 0.13 ²   <0.03 ² complex AMT0068-40 Mabscessus/massiliense  32 ² 1 0.25 ²    0.06 ² complex AMT0119-7 Mabscessus/massiliense  32 ² 1 0.06 ²   <0.03 ² complex AMT0493-2 Mabscessus/massiliense Amikacin resistant   >64 ² 2 0.5 ²     0.125 ²complex AGR1 P. aeruginosa  16 na 1   1 AGR14 P. aeruginosa Multi-drugresistant >64 na 0.5   1 MR14 P. aeruginosa Multi-drug resistant >64 na1   1 SM21 S. maltophilia  64 na 0.25    0.13 AX1 Achromobacter spp.  64na 1   1 AX4 A. xylosoxidans >64 na 0.25    0.5 BC5b B. multivorans (Bcepacia complex) >64 na 0.25   1 BC15 B. cenocepacia (B cepaciacomplex) >64 na 2   4 BC17 B. cepacia (B cepacia complex) >64 na 8   8AU197 B. cenocepacia (B cepacia complex) >64 na 0.5   4

TABLE 4B BisEDT and BisBDT MIC against control bacterial strains ATCC orother strains used to control for drug activity Expected results(acceptable range) mcg/mL (see detailed data for measured MICs with eachassay set-up) (per CLSI M100-S24 or provided by Microbion) StrainSpecies Amikacin Clarithromycin BisEDT BisBDT ATCC 29213 S. aureus 2(1-4) 0.25 (0.12-0.5) 0.25-0.5 (0.13-1) 0.25 (0.12-0.5) ATCC 25922 E.coli 1-2 (0.5-4) na 1 (0.5-2) na

Evaluation of BT Compound Effect on Cytotoxicity on a FullyDifferentiated Human Airway Epithelium (MUCILAIR™)

The aim of this study is to evaluate the potential local toxic effect ofBisEDT on airway epithelium. The project is divided into 2 phases: Study1: Acute Toxicity testing of BisEDT in solution; Study 2: Acute Toxicitytesting of BisEDT as solid.

The assay system used in this study is Epithelix's proprietarytechnology MUCILAIR™. MUCILAIR™ is a fully differentiated andready-to-use 3D model of human airway epithelium, constituted withprimary human epithelial cells freshly isolated from nasal, tracheal orbronchial biopsies. MUCILAIR™ (FIG. 24 ), is not only morphologicallyand functionally differentiated, but can also be maintained in ahomeostatic state for a long period of time (Huang et al., 2009).

MUCILAIR™ is composed of basal cells, ciliated cells and mucus cells.The proportion of these various cell types is preserved compared to whatone observes in vivo (Huang et al., 2011). Moreover the epithelia arestarted from de-differentiated cells. The cells undergo a progressivedifferentiation with time. After 45 days of culture, the epithelia arefully ciliated, secret mucus and are electrically tight (TEER>200Ω·cm²). The activity of the main epithelial ionic channels, such asCFTR, EnaC, Na/K ATPase, is preserved and the epithelia is shown torespond in a regulated and vectorial manner to the pro-inflammatorystimulus, TNF-α (Huang et al., 2011). A large panel of cytokines,chemokines and metalloproteinases has been detected in MUCILAIR™ (e.g.IL-8, IL-6, GM-CSF, MMP-9, GRO-α).

Acute Toxicity Testing of BisEDT in Solution

The aim of this phase is to evaluate the potential acute toxicity ofBisEDT in solution once applied at the apical surface on a 3D model offully differentiated human airway epithelium (MUCILAIR™) after 1, 8, 24and 48 hours exposure.

TABLE 5 patient information Age of the Sex of the Age of the SpecialBatch number patient patients culture comments MD014101 38 years ND 105days Normal donor

TABLE 6 Test material Identification Name Concentrations VehicleSolubility BisEDT (MB-1-B3) 0.001, 0.01, 0.1, 1, 10, 0.5% DMSO in OK 30μM Buffered Saline

BisEDT (MB-1-B3) was applied on the apical surface of MUCILAIR™ during1, 8, 24 and 48 hours (FIG. 13 ). The effect of 6 concentrations wasstudied: 0.001, 0.01, 0.1, 1, 10 and 30 μM. The compound was diluted ina buffered saline solution (NaCl 0.9%-1.25 mM CaCl₂-10 mM HEPES) with0.5% DMSO. 30 μl solution at the selected concentration was applied onthe apical surface of MUCILAIR™. The negative control corresponds to thevehicle solution (0 μM) and untreated cultures. The positive controlcorresponds to 50 μl to 10% Triton X-100 diluted in a buffered salinesolution. The study was run in triplicates.

During the study, inserts were maintained in a CO₂ incubator (37° C., 5%CO₂, 100% humidity). The following end-points were determined:

-   -   Tissue integrity monitoring: Trans-Epithelial Electrical        Resistance (TEER) measurement (quantitative) at D0, D1 and D2.    -   Cytotoxicity monitoring: LDH test (quantitative) at D0, D1 and        D2.    -   Morphology: cellular and tissue integrity examined under        contrast microscope at D0, D1 and D2.

Three days before the experiment, the following quality controls wereperformed on each epithelium:

-   -   Washing: inserts were washed with 200 μl of MUCILAIR™ culture        medium. Washing removes accumulated mucus on the tissue surface        which may interfere with the test.    -   Trans-Epithelial Electrical Resistance (TEER): TEER was measured        to verify that all the selected inserts had tight epithelial        barriers and the tissue itself was not disrupted prior to        application of the test material.    -   Tissue morphology: each insert was inspected under a        conventional inverted microscope to ensure the quality of the        epithelia and determine qualitatively that cilia motion was        visible. The presence of mucus was detected by the refractive        aspect of the apical surface.

Results

Error bars in the following graphs refer to standard error of the mean(SEM). All comparisons are versus the negative control (vehiclesolution, 0 μM).

Before exposure: Each insert was inspected under a conventional invertedmicroscope to insure the quality of the epithelia. The movement of ciliawas clearly visible for all the selected inserts. The presence of mucuswas detected by the refractive aspect of the apical surface.

After exposure: No morphology changes were observed for the vehiclecontrol and all tested concentrations and time points.

Cytotoxicity assessment: FIG. 25 shows the percentage of cytotoxicity(LDH measurement) at 1, 8, 24 and 48 hours exposure. The control (0 μM,vehicle solution) corresponds to a physiological release of LDH (<5%).LDH release was not altered after exposure to BisEDT at all testedconcentrations and at all time-points. Therefore, no toxic effect wasobserved.

Tissue integrity assessment: FIG. 26 shows the monitoring of TEER at D1.It should be noted that TEER is a dynamic parameter that can be affectedby several factors. A decrease of the ionic channel activity can lead toan increase of TEER, and an activation of the ion channels can decreaseTEER values. When an epithelium is damaged, a decrease of TEER would beassociated with an increase of LDH release. BisEDT didn't showsignificant effect on TEER at all tested concentrations and time points.

Acute Toxicity Testing of BisEDT as Solid: 1^(st) Set of Experiments

The aim of this study is to evaluate the potential acute toxicity ofBisEDT as solid (at 0.033, 0.33, 3.33, 33.3, 333 μg/cm²) once applied atthe apical surface on a 3D model of fully differentiated human airwayepithelium (MUCILAIR™) after 1, 8, 24 and 48 hours exposure.

TABLE 7 Tissues (Patient information) Age of the Sex of the Age of theSpecial Batch number patient patients culture comments MD014101 38 yearsND 119 days Normal donor

TABLE 8 Test Material Identification Name Concentrations Vehicle MB-1-B30.033, 0.33, 3.33, 33.3, Dextran powder (C60) 333 μg/cm² Ref:Pharmacosmos 5510 0060 1007

Compound BisEDT was applied on the apical surface of MUCILAIR™. Theeffect of 5 concentrations after 1, 8, 24 and 48 hours exposure wasstudied: 0.033, 0.33,3.33, 33.3, 333 μg/cm². The compound was diluted inDextran powder at the targeted concentration and compressed in order toobtain a tablet. The study was run in triplicates. During the study,inserts were maintained in a CO₂ incubator (37° C., 5% CO₂, 100%humidity). The mucociliary clearance analysis was performed after 1 hourand 24 hours exposure.

Quality control and washing of the apical surface: Three days before theexperiment, the following quality controls were performed on eachepithelium:

-   -   Washing: inserts were washed with 200 μl of MUCILAIR™ culture        medium. Washing removes accumulated mucus on the tissue surface        which may interfere with the test.    -   Trans-Epithelial Electrical Resistance (TEER): TEER was measured        to verify that all the selected inserts had tight epithelial        barriers and the tissue itself was not disrupted prior to        application of the test material.    -   Tissue morphology: each insert was inspected under a        conventional inverted microscope to ensure the quality of the        epithelia and determine qualitatively that cilia motion was        visible. The presence of mucus was detected by the refractive        aspect of the apical surface.

Results

Error bars in the following graphs refer to standard error of the mean(SEM). All comparisons are versus the negative control (Carrier,Dextran).

Before exposure: Each insert was inspected under a conventional invertedmicroscope to insure the quality of the epithelia. The movement of ciliawas clearly visible for all the selected inserts. The presence of mucuswas detected by the refractive aspect of the apical surface.

After exposure: No morphology changes were observed for the non-treatedand the vehicle controls.

-   -   0.033 and 0.33 μg/cm²: No morphological modifications were        observed.    -   3.33 μg/cm²: The tablets applied apically were poorly dissolved        on the epithelia at 24 and 48 h after exposure. Cells were        rounded and opaque at the periphery of inserts. Gradually the        cells became detached from each other. The appearance of culture        medium coming from the basal side on the apical surface was        observed.    -   33.3 and 333 μg/cm²: The tablets applied apically was poorly        dissolved on the epithelia at 8, 24 and 48 h after exposure.        Cells were rounded and opaque on the inserts. Gradually the        cells became detached from each other. The culture medium leaked        to the apical side.

Cytotoxicity assessment: FIG. 27 shows the percentage of cytotoxicity(LDH measurement) at Dl. The control (Dextran) corresponds to aphysiological release of LDH (<5%). No significant effect on LDH releaseat 0.033 and 0.33 μg/cm² at all tested time points. A time and dosedependent, cytotoxicity is observed at 3.3; 33.3 and 333 μg/cm².

Tissue integrity assessment: FIG. 28 shows the monitoring of TEER at D1.It should be noted that TEER is a dynamic parameter that can be affectedby several factors. A decrease of the ionic channel activity can lead toan increase of TEER, and an activation of the ion channels can decreaseTEER values. When an epithelium is damaged, a decrease of TEER would beassociated with an increase of LDH release. No significant effect wasobserved on TEER at 0.033 and 0.33 μg/cm² at all tested time points.After 1 hour exposure, no significant effect on TEER is observed at alltested doses. Loss of tissue integrity is observed at 3.33, 33.3 and 333μg/cm² after 8, 24 and 48 H exposure.

Acute Toxicity Testing of BisEDT as Solid: 2^(nd) Set of Experiments

The aim of this phase is to evaluate the potential acute toxicity ofBisEDT as solid (at 1 μg/cm²) once applied at the apical surface on a 3Dmodel of fully differentiated human airway epithelium (MUCILAIR™) after1, 8, 24 and 48 hours exposure.

TABLE 9 Patient Information Age of the Sex of the Age of the SpecialBatch number patient patients culture comments MD014101 38 years ND 125days Normal donor

TABLE 10 Compound Information Identification Name Concentrations VehicleMB-1-B3 1 μg/cm² Dextran powder (C60) Ref: Pharmacosmos 5510 0060 1007

Testing Strategy and Protocol: Compound BisEDT was applied on the apicalsurface of MUCILAIR™. The effect of 1 μg/cm² concentrations after 1, 8,24 and 48 hours exposure was studied. The compound was diluted inDextran powder at the targeted concentration and compressed in order toobtain a tablet. The study was run in triplicates. During the study,inserts were maintained in a CO² incubator (37° C., 5% CO2, 100%humidity). The mucociliary clearance analysis was performed after 1 hourand 24 hours exposure.

Quality control and washing of the apical surface: Three days before theexperiment, the following quality controls were performed on eachepithelium:

-   -   Washing: inserts were washed with 200 μl of MUCILAIR™ culture        medium. Washing removes accumulated mucus on the tissue surface        which may interfere with the test.    -   Trans-Epithelial Electrical Resistance (TEER): TEER was measured        to verify that all the selected inserts had tight epithelial        barriers and the tissue itself was not disrupted prior to        application of the test material.    -   Tissue morphology: each insert was inspected under a        conventional inverted microscope to ensure the quality of the        epithelia and determine qualitatively that cilia motion was        visible. The presence of mucus was detected by the refractive        aspect of the apical surface.

Results

Error bars in the following graphs refer to standard error of the mean(SEM). All comparisons are versus the negative control (CarrierDextran).

Morphology

Morphology, Before exposure: Each insert was inspected under aconventional inverted microscope to insure the quality of the epithelia.The movement of cilia was clearly visible for all the selected inserts.The presence of mucus was detected by the refractive aspect of theapical surface.

Morphology, After exposure: No morphology changes were observed for thenon-treated and the vehicle controls. 1 μg/cm²: after 1 hour and 8 hourexposure, no important morphological changes were observed. After 24 hexposure, the epithelial cells on 2 inserts out of 3 were dead. After 48h the epithelial cells on all 3 inserts were dead.

Cytotoxicity assessment: FIG. 29 represents the percentage ofcytotoxicity (LDH measurement) at D1. The control (Dextran) correspondsto a physiological release of LDH (<5%). No significant effect on LDHrelease at 1 μg/cm² after 1 hour exposure. A time- and dose-dependentcytotoxicity is observed at 8, 24 and 48 h after exposure.

Tissue Integrity Assessment: FIG. 30 represents the monitoring of TEERat D1. It should be noted that TEER is a dynamic parameter that can beaffected by several factors. A decrease of the ionic channel activitycan lead to an increase of TEER, and an activation of the ion channelscan decrease TEER values. When an epithelium is damaged, a decrease ofTEER would be associated with an increase of LDH release.

After 1 hour exposure, no significant effect on TEER was observed at 1μg/cm². Loss of tissue integrity is observed at 1 μg/cm² after 8, 24 and48 H exposure. After 48 H exposure, the epithelia were no more tight.

CONCLUSIONS

The aim of this study is to evaluate the potential local toxic effect ofBisEDT on airway epithelium in liquid or solid solution. To evaluatepotential effects of BisEDT exposure on fully differentiated human nasalepithelia cultured at the air-liquid interface, two sets of experimentswere conducted namely BisEDT in liquid solutions or BisEDT as solid.

In general, within the presented study, BisEDT compound in liquidsolution showed no local toxicity on the airway epithelium at differenttested concentrations (0.001, 0.01, 0.1, 1, 10, 30 μM) and time exposure(1, 8, 24, and 48 hours). No effect on the morphology of the epithelia,on TEER and LDH release were observed.

Similar results were obtained when the compound was applied as solid atlow doses (0.033 and 0.33 μg/cm² for 1, 8, 24, and 48 hour exposures).However, at 1 and 3.33 μg/cm², BisEDT induces cytotoxicity at 24 h and48 h exposure, demonstrated by an increase of LDH release and areduction of the TEER value. For higher concentrations (33.3, 333μg/cm²), strong toxicity was observed with a high amount of LDHreleased, a very low TEER values and important morphological changes.

REFERENCES

Huang, S; Caul-Futy, M; “A novel in vitro cell model of the human airwayepithelium” 3R-Info-Bulletin No. 41, October 2009.

Huang, S., Wiszniewski, , & Constant, S. (2011). The use of in vitro 3Dcell models in drug development for respiratory diseases. Drug Discoveryand Development—Present and Future

Example 6 Sputum Studies

Bacterial killing curves with BisEDT and BisBDT were performed in thepresence of three cystic fibrosis patient sputum in order to determinewhether the test compounds are potentially inactivated by sputum. Theassay is described in King P, Lomovskaya O, Griffith D C, Burns J L,Dudley M N. In vitro pharmacodynamics of levofloxacin and otheraerosolized antibiotics under multiple conditions relevant to chronicpulmonary infection in cystic fibrosis. Antimicrob Agents Chemother,54:143-8, 2010.

Sputum was collected from cystic fibrosis (CF) patient volunteerswithout recent antibiotic exposure after appropriate IRB approval.Sputum was sterilized by UV irradiation and sterilization was confirmedby culture. An overnight culture of Pseudomonas aeruginosa strain PA01was used to inoculate fresh cultures in cation-adjusted Mueller-Hintonbroth or cation-adjusted Mueller-Hinton broth plus 10% CF patientsputum. Drugs were added to individual culture tubes with and withoutsputum at the following final concentrations:

BisEDT: 0.1 μg/mL, 2 μg/mL, and 20 μg/mL

BisBDT: 0.1 μg/mL, 4 μg/mL, and 20 μg/mL

Tobramycin at 1 μg/mL was used as a comparator drug control known to bepartially inactivated by patient sputum. Cultures were incubated withshaking at 37° C. and aliquots were removed every hour for quantitationof colony forming units per mL (CFU/mL) by serial dilution and platingon tryptic soy agar for a total of 6 hours. CFU were counted afterincubation of the plates overnight at 37° C.

The controls for this assay performed as expected. The growth of PA01was not obviously inhibited or enhanced by the addition of sterilepatient sputum in the absence of additional drug to bacterial cultures(FIG. 31 , closed and open circles). As shown, sputum partially inhibitsthe bacterial killing activity of tobramycin, with approximately 0.5-1log CFU/mL higher at most time points in cultures with sputum comparedto cultures without sputum.

The bactericidal activity of BisEDT appears to be partially inhibited byCF patient sputum based on this assay (FIG. 32 ). BisEDT is notbactericidal against PA01 at 0.1m/mL. With BisEDT at 2 μg/mL, theaddition of sputum reduces killing by approximately 1-2 log CFU/mL at 3to 6 hours. When the concentration of BisEDT is further increased to 20μg/mL the inhibition of bacterial killing by sputum is less pronounced,with killing in the presence of sputum lagging by only 0.5-1 log CFU/mLbehind cultures without sputum at early time points (1-3 hours).Eventually, with this highest dose tested, bacteria in the culture withsputum is reduced below the limit of detection 1 hour earlier than thesample without sputum, suggesting that at higher drug concentrationsinhibition of BisEDT by sputum is largely overcome.

Similarly, the bactericidal activity of BisBDT appears to be partiallyinhibited by CF patient sputum (FIG. 33 ). BisBDT at 0.1 μg/mL is notbactericidal against PA01. In the absence of sputum, 4 μg/mL BisBDTdemonstrated very slow bactericidal activity against PA01, with killingof only about 1 log over the 6 hour assay; cultures with sputumdemonstrated approximately 0.5-0.8 log CFU/mL more surviving at 3-6hours. At the highest concentration of BisBDT tested, 20 μg/mL, there isan initial lag in killing in PA01 at 1-2 hours with the addition ofsputum, but both samples with and without sputum are sterilized belowthe limit of detection by 5 hours.

Both compounds BisEDT and BisBDT are bactericidal against Pseudomonasaeruginosa strain PA01 in this assay, and this bactericidal activity ispartially inhibited in the presence of CF patient sputum. This partialinhibition of bactericidal activity can be overcome by increasedconcentration of the test compounds. Thus, a higher concentration ofbismuth-thiol compound maybe needed in areas where sputum is presentcompared to bodily compartments without sputum.

Example 7 In Vitro Activity of Bismuth Thiols and Comparators AgainstHaemophilus Influenza Clinical Isolates

In this Example, the activity of three Bismuth thiol compounds areevaluated against Haemophilus influenzae, a prevalent pathogen ofrespiratory disease including pneumonia, otitis media, conjunctivitis,and meningitis.

Test and Control Agents: The test compounds (BisEDT, and analogsBisBDT/PYR and BisEDT/PYR) were shipped from to Micromyx and stored at−20° C. until assayed. The solvent for all test compounds was 100% DMSO,the stock concentration was 2560 μg/mL, and the range tested was 64-0.06μg/mL. All stock solutions were allowed to stand for at least one hourprior to use to auto-sterilize.

Comparator drugs were provided by Micromyx. Suppliers, lot numbers,diluent, stock concentrations, and test ranges were as follows:

Concentration of Stock Test/Control Solution Test Range Agents SupplierLot No. Diluent (μg/mL) (μg/mL) Azithromycin USP JOI240 DMSO 256064-0.06 Ampicillin Sigma BCBF0407V Sorenson 1280 32-0.03 Buffer pH 7.5Cefuroxime Sigma 031M0823V dH₂O 2560 64-0.06 Levofloxacin SigmaBCBC2112V DMSO 2560 64-0.06  1-0.001

Test Organisms

The test organisms were maintained frozen at −80° C. The organisms wereoriginally acquired from the American Type Culture Collection or fromclinical laboratories. The isolates were sub-cultured on Chocolate Agarplates (Remel; Lenexa, Kans.) and incubated overnight at 35° C. with 5%CO₂ . H. influenzae ATCC 49247 was tested for the purposes of qualitycontrol for the comparator compounds.

Minimal Inhibitory Concentration (MIC) Assay Media: The medium employedfor the broth microdilution MIC assay was Haemophilus Test Medium (HTM;Remel; Lenexa, Kans.; Catalog No. R112380; Lot No. 056737) asrecommended by the Clinical Laboratory Standards Institute (CLSI; 1).

Broth Dilution Minimal Inhibitory Concentration (MIC) Assay Procedure:MIC values were determined using a broth microdilution method asrecommended by CLSI (1, 2). Automated liquid handlers (Multidrop 384,Labsystems, Helsinki, Finland; Biomek 2000 and Biomek F/X, BeckmanCoulter, Fullerton CA) were used to conduct serial dilutions and makeliquid transfers.

The wells of a standard 96-well microdilution plate (Costar 3795;Corning Inc.; Corning, N.Y.) were filled with 150 μL of the appropriatesolvent in columns 2-12 on the Multidrop 384. This plate was used toprepare the drug “mother plate” which provided the serial drug dilutionsfor the replicate “daughter plates”. The Biomek 2000 was used totransfer 150 μl of each stock solution from the wells in Column 1 of themother plate to make ten subsequent 2-fold serial dilutions. The wellsof Column 12 contained no drug and were the organism growth controlwells. Each mother plate has the capacity to create a total of 15daughter plates.

The daughter plates were loaded with 185 μL of HTM using the Multidrop384. The wells of the daughter plates ultimately contained 185 μL ofHTM, 5 μL of drug solution, and 10 μL of inoculum. The daughter plateswere prepared on the Biomek F/X instrument which transferred 5 μL ofdrug solution from each well of the mother plate to each correspondingwell of each daughter plate in a single step.

A standardized inoculum of each organism was prepared per CLSI methods(1). The inoculum for each organism was dispensed into sterilereservoirs divided by length (Beckman Coulter), and the Biomek 2000 wasused to inoculate the plates. Daughter plates were placed on the Biomek2000 work surface reversed so that inoculation takes place from low tohigh drug concentration. The Biomek 2000 delivered 10 μL of standardizedinoculum into each well. These dilutions yielded a final cellconcentration in the daughter plates of approximately 5.0×105colony-forming-units/mL.

Plates were stacked three high, covered with a lid on the top plate,placed in plastic bags, and incubated at 35° C. for approximately 24 hr.Following incubation, the microplates were removed from the incubatorand viewed from the bottom using a plate viewer. An un-inoculatedsolubility control plate was observed for evidence of drugprecipitation. The MIC was read and recorded as the lowest concentrationof drug that inhibited significant visible growth of the organism.

Results

Examination of the un-inoculated drug solubility plates revealed noprecipitation of any of the evaluated agents over the testedconcentration range. Against H. influenzae ATCC 49247, evaluatedcomparators had MICs within the acceptable range for quality controlestablished by CLSI (2) where applicable.

All Bismuth Thiol test compounds were active against all evaluated H.influenzae strains regardless of resistance to other agents, includingisolates which were beta-lactamase negative ampicillin-resistant(BLNAR). The MICs of BisEDT and analogs BisEDT/PYR and BisBDT/PYR wereall at or below the lowest concentration evaluated in the study (<0.06μg/mL). This activity was greater than azithromycin (MIC₅₀ and MIC₉₀ of2 μg/mL), ampicillin (MIC₅₀ of 2 μg/mL and MIC₉₀ of 4 μg/mL), andcefuroxime (MIC₅₀ of 4 μg/mL and MIC₉₀ of 16 μg/mL). Levofloxacin, whichwas tested down as low as 0.001 μg/mL for purposes of quality controlhad an MIC₅₀ and MIC₉₀ of 0.015 μg/mL. As the Bismuth Thiol compoundswere tested from 0.06-64 μg/mL in this study, and their MICs were <0.06μg/mL, it is not known whether their activity exceeds that oflevofloxacin which was generally had MICs of 0.015 μg/mL.

This study demonstrated a high degree of potency for the tested BismuthThiol compounds against H. influenzae at levels exceeding that ofampicillin, azithromycin, and cefuroxime. This activity illustrates thatH. influenzae is included as part of the broad spectrum of activityshown in vitro for this class of novel therapeutic agents.

REFERENCES

1.) Clinical and Laboratory Standards Institute. Methods for DilutionAntimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;Approved Standard—Ninth Edition. Clinical and Laboratory StandardsInstitute document M07-A9 [ISBN 1-56238-784-7]. Clinical and LaboratoryStandards Institute, 940 West Valley Road, Suite 1400, Wayne, Pa.19087-1898 USA, 2012.

2.) Clinical and Laboratory Standards Institute. Performance Standardsfor Antimicrobial Susceptibility Testing; Twenty-Second InformationalSupplement. CLSI document M100-S22 [ISBN 1-56238-786-3]. Clinical andLaboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,Pa. 19087 USA, 2012.

Example 8 In Vivo Studies

Objectives: The primary objective of this study is to assess thetolerance of BisEDT (Dalton Pharma Services; Lot #ED268-1-11-01 storedat room temperature) following nose only inhalation exposure in F344rats. Animals will be exposed to differing concentrations (low, middle,high) for up to 180 minutes. Blood will be collected at predeterminedtimepoints to analyze for systemic presence of BisEDT. At the conclusionof the study (24 hr post exposure) animals will be euthanized andundergo necropsy where lungs will be lavaged and collected forpotential, future analysis. Additional respiratory tract related tissuessuch as the nasal cavity, larynx, pharynx, nasopharynx, trachea,bronchus, and carina will also be collected. Lavage fluid will beanalyzed for clinical chemistry and hematology parameters including cellcounts and differentials. Liver, kidney, esophagus, stomach, smallintestine, and large intestine will also be collected for potential,future analysis.

Animals: Male and female F344 rats provided by Charles River Labs,Wilmington, MA were used in this study. The rats were approximately 7-9weeks old at the time of arrival and 8-10 weeks at study initiation.Body weights of individual animals were ±20% of the group mean. Animalswill be uniquely identified by numeric tail markings (made withindelible ink such as a Sharpie®) for body weights, randomization, andtreatment administration. Color coded cage cards will also be placed onthe cages. A total of forty (40) male and female F344 rats (20 M/20 F)(including spares) were ordered for the study. Thirty six (36) animalswere randomized into 3 study groups each consisting of 12 animals pergroup (6 M/6F). The remaining 4 animals (2 M 12 F) were spares. Uponremoval from quarantine, thirty six (36) rats will be randomized into 3study groups each consisting of 12 animals (6 M/6 F) per group by bodyweight stratification. Unused spares will be either euthanized orconveyed to another approved study protocol. Prior to the start ofexposures, animals will be conditioned to nose-only exposure tubes.

Animal Husbandry: Animals will be housed for a minimum of 7 days, up to2 per cage in polycarbonate shoebox cages with Alpha Dri or hardwoodchip bedding. Caging and bedding were autoclaved. Prior to injection,animals were introduced at least once to restraint tubes that will beused for tail vein dosing. Animal feed was 2016C Harlan Global CertifiedRodent Chow, (Harlan Tekland, Madison, Wis.), unlimited access exceptduring study procedures. Each batch of feed wass analyzed forcontaminants by the manufacturer and will be used within themanufacturer's designated shelf-life. Animals were provided municipalwater (filtered at 5, 1, and 0.2 μm), unlimited access except duringstudy procedures. Only healthy animals were used in this study. Alaboratory animal veterinarian or designee visually examined the animalsbefore release from quarantine.

Environmental Conditions: The targeted conditions for animal roomenvironment and photoperiod will be as follows: Temperature: 18-26° C.;Humidity: 30-70%; Light Cycle: 12-h. Light, humidity, and temperatureexcursions are defined as a sustained reading that falls out of thespecified range for more than 3 hours.

Experimental Design: The experimental design for this study was: Group1: Low dose; inhalation; 6 males and 6 females; blood collection at 0.5,2 hr and 8 hr post exposure and 24 hr (terminal); and necropsy at 24hours-post inhalation. Group 2: Mid dose; inhalation; 6 males and 6females; blood collection at 0.5, 2 hr and 8 hr post exposure and 24 hr(terminal); and necropsy at 24 hours-post inhalation. Group 3: highdose; inhalation; 6 males and 6 females; blood collection at 0.5, 2 hrand 8 hr post exposure and 24 hr (terminal); and necropsy at 24hours-post inhalation. Animals were weighed and randomized into studygroups following the quarantine period. Animals were broken into 3groups each consisting of 6 M and 6 F per group. Groups were exposed tothree different dose levels of BisEDT. The initial exposure was to aformulation concentration of 100 mg/mL for an exposure time of 60minutes; based on method development this is expected to result in adose of 3 mg/kg. The maximum duration for exposures was 180 minutes andthe maximum formulation concentration was 100 mg/mL.

Following exposure, non-terminal blood collections were performed on 2animals per sex at 0.5, 2, and 8 hours post exposure. At the conclusionof the study (24 hr post exposure) animals were euthanized and underwentnecropsy. The right lung was lavaged, flash frozen, and collected forpotential, future analysis. The left lung was flash frozen forpotential, future analysis. Additional respiratory tract related tissuessuch as the nasal cavity, larynx, pharynx, nasopharynx, trachea,bronchus, and carina were collected. Lavage fluid was analyzed forclinical chemistry and hematology parameters including cell counts anddifferentials. Liver, kidney, esophagus, stomach, small intestine, andlarge intestine were collected for potential, future analysis. Theseorgan tissue samples were flash frozen and stored pending studycompletion. The lavage fluid were analyzed for clinical pathology andhematology (including cell counts and differentials). A maximum wholeblood collection was also collected at necropsy. Blood samples wereanalyzed for clinical chemistry and hematology as well as an aliquotsnap frozen for potential analysis for BisEDT.

Inhalation Exposure: The initial exposure of BisEDT(Bismuth-1,2-ethanedithiol) was formulated as a solution at 100 mg/mL insuspension in 0.5% TWEEN 80®, 10 mM sodium phosphate, pH 7.4, in NaCl(adjusted to approximately 300 mOsm). Aerosols were generated with acommercial compressed air jet nebulizer, PAD LC PLUS®, operated with aninlet pressure of 20 psi. A schematic is shown in FIG. 1 . The aerosolswere transitioned into a rodent nose-only inhalation exposure system.The exposure system was operated with an inlet air flow of −5.2 L/minand an exhaust air flow of −5 L/min. This resulted in 0.31 L/min to eachport which is slightly greater than 1.5×the respiratory minute volume ofa rat.

Exposure Concentration and Particle Size Monitoring: Aerosolconcentration was monitored at the breathing zone by collection onto aGF/a filter. The filters were analyzed via differential mass and andother methods known in the art. Aerosol particle size was measured usinga TSI Aerodynamic Particle Sizer (Model 3321, TSI, Inc., Shoreview,Minn.) or an In-Tox Mercer 2.0 L/min cascade impactor.

Observations and measurements: Observations were documented in Provantisdatabase or the Animal Management System (AMS, LRRI, Albuquerque).

Clinical Observations and Mortality/Morbidity: Detailed clinicalobservations were recorded starting on dose day with observationsrecorded prior to exposure, during exposure, after exposure as theanimals are returned to their home cages, and in the afternoonpost-exposure. Observations will be recorded according to a standardlexicon (SOP TXP-1532—Pharmacologic and Toxicologic Observations ofExperimental Animals). General observations include but are not limitedto apnea, labored breathing, malaise, marked nasal discharge, etc.Special attention was paid to clinical signs associated with therespiratory tract. Animals showing severe signs of distress wereeuthanized immediately at the discretion of the Study Director inconsultation with veterinary staff. Examinations were also orientedtoward (1) identifying dead, weak, or moribund animals, and (2)documenting the onset and progression of any abnormal clinical signs.Moribund or dead animals were necropsied as soon as possible after beingfound but in no case later than 16 hours after being found.

Body Weights: All animals were weighed after release from quarantine andthat weight will be the pre-study body weight used to randomize animalsinto dose groups. Body weights were collected in the morning prior toexposure and again at necropsy.

Blood Collection for Clinical Chemistry and Hematology: Blood samplesfor hematology and clinical chemistry were collected during necropsy.For Complete Blood Count (CBC) with absolute differentials, whole blood(target 0.5 mL) was collected and placed into tubes containingtripotassium ethylenediaminetetraacetate (K₃EDTA) as an anticoagulant.Hematology samples was analyzed by automated (ADVIA™ 120 HematologySystem, Siemens Medical Solutions Diagnostics, Tarrytown, N.Y.)analyses. The parameters for hematology are: Red Blood Cell Count (RBC)10⁶/μL; Hemoglobin (HGB) g/dL; Hematocrit (HCT) %; Mean CorpuscularVolume (MCV) fL; Concentration (MCHC) g/dL; Mean Corpuscular Hemoglobin(MCH) pg; Platelet Count (PLT) (10³/μL); Percent Reticulocytes (RETIC) %RBC; White Blood Cell Count (WBC) 10³/μL; Neutrophils (PMN) 10³/μL;Lymphocytes (LYM) 10³/μL; Monocytes (MONO) 10³/μL; Eosinophils (EOS)10³/μL; Basophils (BASO) 10³/μL; Large Unstained Cells (LUC) 10³/μL.

For clinical chemistry analyses, whole blood (≥0.5 mL) was placed intoserum separator or clot tube for centrifugation and separation intocellular and serum fractions. Serum samples were analyzed on a HitachiModular Analytics Clinical Chemistry System (Roche Diagnostics,Indianapolis, IN). The clinical chemistry parameters measured orcalculated are: Alanine Aminotransferase (Alanine Transaminase)-Serum(ALT) IU/L; Albumin (ALB) g/dL; Aspartate Aminotransferase (AspartateTransaminase)-Serum (AST) IU/L; Bilirubin (Total) (BILI-T) mg/dL; BloodUrea Nitrogen (BUN) mg/dL; Calcium (CA) mg/dL; Chloride (Serum) (CL-S)mmol/L; Cholesterol (Total) (CHOL) mg/dL; Creatinine (Serum) (CRE-S)mg/dL; Glucose (GLU) mg/dL; Gamma Glutamyltransferase (GGT) IU/L;Alkaline Phosphatase (ALP) IU/L; Phosphate (PHOS) mg/dL; Potassium(Serum) (K-S) mmol/L; Protein (Total) (TP) g/dL; Sodium (Serum) (NA-S)mmol/L; Triglycerides (TRIG) mg/dL; Albumin/Globulin (A/G) no units;Blood Urea Nitrogen/Creatinine (BUN/CRE) no units; Globulin (GLOBN)g/dL.

Hematology and clinical chemistry evaluations were performed on allstudy animals for which adequate sample volumes are obtained and forwhich no analytical problems are encountered. If target collectionvolumes are not obtained or if evaluations are not performed, a reasonand notation will be included in the raw data. The remaining bloodsamples or serum will be discarded after the analyses.

Blood Collection for Bioanalytical Analysis: Following exposure,non-terminal blood collections were performed on 2 animals per sex at0.5, 2, and 8 hours post exposure. Approximately 1 mL of systemic wholeblood was collected by jugular vein into tubes containing K3EDTA as ananticoagulant. The tubes were flash frozen with liquid nitrogen storedwithout processing at −70 to −90° C. until shipping for analysis usingICP-MS assay for quantitation of bismuth as a surrogate for BisEDT.

Euthanasia and necropsy: Animals were fasted overnight prior toscheduled necropsy (24 hr post exposure). At scheduled necropsy or incases of morbidity, animals were euthanized by intraperitoneal injectionof an overdose of a barbiturate-based sedative. Detailed grossnecropsies were performed on all animals (found dead, moribund, orscheduled necropsy) and consisted of a complete external and internalexamination including body orifices and cranial, thoracic, and abdominalorgans and tissues. All gross findings will be recorded in descriptiveterms. Whole blood was collected for bioanalytical analysis, hematology,and clinical chemistry at necropsy. Lungs collected and weighed. Theleft lobe were tied off and flash frozen for potential, future analysis.The right lobes were lavaged 3× using 4 mL/lavage of phosphate bufferedsaline (PBS). After lavage is complete the right lobes were flash frozenin liquid nitrogen for potential, future analysis.

This Example describes the results of BisEDT single-dose rat PK studiescomparing inhalation, IV, and oral dosing. The primary takeaways arethat (1) BisEDT remains in lung tissue after inhalation dosing with ahalf-life of about 4 days (FIG. 34 ); (2) there are very low, butsustained and measurable blood concentrations after oral dosing; (3) IVdosing appears to follow a biphasic pattern with initial distributioninto tissues for 18 hours, followed by a slow systemic eliminationphase; (4) BisEDT does not appreciably partition into lung tissue afterIV or oral dosing—no lung levels after oral and low lung levels detectedafter IV dosing (<5% vs inhalation group) and levels dropped rapidlywith about 24 hour half-life. This indicates that systemic BisEDT doesnot partition into lung tissue and that lung levels measured afterinhalation dosing are due to deposited drug particles on the apicalsurface; (5) after inhalation dosing, there is sustained, moderate,blood exposure with relatively stable concentrations across time,indicating the drug in the lungs is acting as a depot; (6) BisEDT wastolerated at 100 μg/kg inhalation and IV and 250 μg/kg oral. Based onthese data, low doses given daily or every other day are likely toprovide very stable drug levels in tissue and blood (small differencesbetween min and max). If the safety margin is demonstrated to be largeenough during GLP toxicology and/or clinical studies, it is possible tolengthen the dosing interval and the increase the dose accordingly.Increasing the dose and interval leads to larger fluctuations in tissueand blood levels between peak (after dosing) and trough levels (prior todosing).

Based on existing in vivo and in vitro toxicology data as well as rat PKand in vitro MIC data, BisEDT suspension for inhalation can be reliablyadministered at doses providing efficacious lung levels that aretolerated, and it is therefore a viable clinical development candidate.As noted above, BisEDT remains in lung tissue long after inhalationdosing with a half-life of about 4 days (FIG. 35 ). 24 hours aftersingle inhalation dose 4,093 ng/g was measured in the lung tissue (whichequates to 123 μg/mL in lung fluid) and 4 days (96 hrs) after singledose 2,600 ng/g in lung tissue (which equates to 78 μg/mL in lungfluid). Without being bound by any particular theory, it is believedBisEDT's low solubility provides slow dissolution and long exposure onlung surface fluids with limited systemic exposure via diffusion throughlung epithelium. The lung appears to act as a depot for slow systemicexposure with about 10×lower systemic levels than seen in the lung. Theterminal phase of the IV data appears to show slow elimination afterinitial tissue distribution (FIG. 36 ).

Tables 11 and 12 below show whole blood BisEDT versus time data and lungBisEDT concentration at sacrifice time data respectively. These resultsare also shown graphically in FIGS. 37 and 38 .

TABLE 11 Whole Blood BisEDT versus Time Data Mean BisEDT Dose NominalConc (μg/kg) Time (hr) (ng/mL) STDEV 3000 1 485 244 3000 2 2504 547 30008 6570 1729 3000 24 20525 4907 47 8 114 64 47 12 98 35 47 24 282 84 4730 373 201 400 8 1701 542 400 12 1252 368 400 24 2700 607 400 30 27381028 124 8 247 3 124 12 187 78 124 24 571 93 124 30 668 239 0 30 0 N/A 030 0 N/A 0 30 0 N/A

TABLE 12 Lung BisEDT Conc. at Sacrifice Mean BisEDT Dose Sac time Conc(μg/kg) (hr) (ng/g) STDEV 3000 24 82.3 8.7 400 30 12.1 4 124 30 5.9 0.947 30 4.8 1.1 0 30 0 N/A

The efficacy of BisEDT in reducing pulmonary bacterial burden associatedwith pulmonary infection in a rat model of pulmonary Pseudomonasaeruginosa infection was evaluated. The results follow.

Aerosol concentration: The results of aerosol concentration (gravimetricand chemistry) are presented in Table 13. No BisEDT was detected bychemical analysis of the vehicle, as expected. Gravimetric analysis islisted. The positive control (Tobramycin) concentration was determinedgravimetrically and all exposure atmospheres varied by less than 10%through both cohorts and all exposure days. The test articleconcentration, determined by chemical analysis, varied by less than 5.1%for all exposure cohorts and days and was 11.5 μg/L.

TABLE 13 Average concentration of exposure atmospheres AnalysisConcentration Group Method Average RSD Vehicle Gravimetric 0.11 ± 0.02mg/L 14.8% Tobramycin Gravimetric 1.26 ± 0.10 mg/L 8.3% BisEDT Chemical11.5 ± 0.6 μg/L 5.1%

Dose Delivered: Tables 14 and 15 describe the calculated presented andtheoretical deposited doses delivered to the animals in this study. Thepresented dose is defined as the total inhaled amount of material,comprising material deposited in the sinuses, throat, oropharyngealregion, lung, as well as exhaled material. The theoretical depositeddose, or amount of material that is actually deposited on the surface ofthe lung, is considered to be 10% of the presented dose in rats (InhalToxicol. 2008 October; 20(13):1179-89. doi: 10.1080/08958370802207318,which is hereby incorporated by reference in its entirey). Each animal'scurrent body weight and exposure condition were factored into theequations above for dose determination. Group 3 and Group 4 exposureswere 13 minutes and 30 minutes in duration, respectively. The vehiclefilters were analyzed chemically for BisEDT and all filters were belowdetection limits. The positive control (tobramycin) filters wereanalyzed gravimetrically and the average presented dose for all cohortsand days was 29.0 mg/kg, higher than the 20 mg/kg value specified in thestudy protocol. The average presented doses for the low and highconcentration groups of BisEDT were 0.114 mg/kg and 0.264 mg/kg,respectively. The study protocol called for doses of 0.1 mg/kg and 0.25mg/kg for those groups. Details of doses received by exposure groups byday and cohort are listed in Tables 14 and 15 below.

TABLE 14 Presented Dose Summary Cohort 1 Dose Cohort 2 Dose Group Day(mg/kg) (mg/kg) Vehicle 0 BDL BDL Vehicle 2 BDL BDL Vehicle 4 BDL BDLTobramycin 1 31.0 ± 3.4  28.9 ± 1.8  Tobramycin 2 29.0 ± 1.9  27.9 ±0.8  Tobramycin 3 27.9 ± 0.7  29.3 ± 1.8  Tobramycin 4 29.3 ± 1.7  28.5± 2.3  BisEDT 0 0.121 ± 0.002 0.110 ± 0.002 (0.1 mg/kg) BisEDT 2 0.120 ±0.002 0.120 ± (0.1 mg/kg) 0.002^(Error! Bookmark not defined.) BisEDT 40.101 ± 0.002 0.114 ± 0.002 (0.1 mg/kg) BisEDT −1 0.263 ± 0.005 0.264 ±0.003 0.25 mg/kg

TABLE 15 Theoretical Deposited Dose Summary Group Day Cohort 1 DoseCohort 2 Dose Vehicle 0 BDL BDL Vehicle 2 BDLBDL^(Error! Bookmark not defined.) Vehicle 4 BDL BDL Tobramycin 1  3.1mg/kg  2.9 mg/kg Tobramycin 2  2.9 mg/kg  2.8 mg/kg Tobramycin 3  2.8mg/kg  2.9 mg/kg Tobramycin 4  2.9 mg/kg  2.9 mg/kg BisEDT 0 12.1 μg/kg11.0 μg/kg (0.1 mg/kg) BisEDT 2 12.0 μg/kg 12.0μg/kg^(Error! Bookmark not defined.) (0.1 mg/kg) BisEDT 4 10.1 μg/kg11.4 μg/kg (0.1 mg/kg) BisEDT −1 26.5 μg/kg 26.4 μg/kg 0.25 mg/kg

Particle Size Distribution: Particle size distributions (FIGS. 38-40 )for each exposure condition was determined using an In-Tox MercerImpactor. FIG. 39 shows the aerosol size distribution of BisEDT measuredduring the study. Table 16 provides summaries of particle sizedistributions (PSD) and geometric standard deviations (GSD) for eachexposure type.

TABLE 16 Summary of size distributions for different exposure conditionsExposure Conditions MMAD (μm) GSD (μm) Vehicle 1.26 1.81 Tobramycin 2.811.87 BisEDT 1.54 1.83

FIG. 42 shows rat efficacy figures showing cumulative (total)administered dose (lung deposited) at days 3 and 5. As can be seen fromthis figure, the mass ratio of delivered drug is staggering compared toTobi.

Conclusion: Animals were divided into four experiment inhalationaltreatment groups: 1) Group 1, a negative control group treated withsaline on Days 0, 2, and 4; 2) Group 2, a positive control group treatedwith tobramycin (target of 20 mg/kg, BID, Days 1-4); 3) Group 3, BisEDT(target of 0.1 mg/kg, QD, Days 0, 2, 4); and Group 4, BisEDT (target of0.25 mg/kg, once, Day-1). Animals were further divided into two cohorts,separated by one day, to accommodate the challenge and treatment of thenumber of animals. All animals were exposed per protocol except Group 1and 3, cohort 2. These animals were exposed on Days 0, 1, and 4 insteadof on Days 0, 2, 4.

BisEDT was not detected in animals receiving saline (negative control).Animals treated twice daily (8 doses) with tobramycin received anaverage dose of 29.0 mg/kg. This dose represents 145% of the targeteddose. As tobramycin is used as a positive control to ensure the modelfunction rather than as a direct comparison, or as a competitor, for theactivity of the test article, the increased dose does not impact thestudy. Animals treated with three targeted doses of 0.1 mg/kg BisEDTreceived an average of 0.114 mg/kg which represents 114% of the targeteddose. For Group 3 (targeted dose of 0.1mg/kg), cohort 1 and 2 averagedoses for each day were, 0.114 and 0.115, respectively. An unpairedt-test (GraphPad Prism 5.0) comparing average dose over the threetreatment days demonstrated the differences between cohorts to not besignificant (p-value=0.93). Animals which received the singleprophylactic targeted dose of 0.25 mg/kg received nearly identical dosesof 0.263 mg/kg (cohort 1) and 0.264 mg/kg (cohort 2) of BisEDT, or 105%and 106%, respectively of the targeted dose.

The particles generated for exposure were considered respirable. Thepositive control (tobramycin) average MMAD of 2.81 μm with a GSD 1.87μm. Aerosol particle MMAD of negative control (saline) treatment andtest article (BisEDT) treatment were 1.26 μm and 1.54 μm with a GSD of1.81 and 1.83, respectively.

Example 9 Study of BisEDT Following Face Mask Inhalation in Beagle Dogs

The objective of the study is to determine the maximum tolerated dose(MTD) or maximum feasible dose (MFD) of inhaled bismuth ethanedithiol(BisEDT) after face-mask inhalation exposure (up to 60 min of exposure)in male and female beagle dogs. Animals will be monitored for up to 14days following the first day of exposure and blood will be collected atpredetermined timepoints to analyze the pharmacokinetics of BisEDT. Inaddition, a coagulation panel will be conducted prior to necropsy. Atthe conclusion of the study animals will be euthanized and undergonecropsy during which the respiratory tract will be harvested forbioanalytical and histopathological analysis. Results obtained willinform a follow up repeated dose GLP study. This test article iscurrently being developed as a novel formulation of a broad spectrumantimicrobial/antibiofilm agent, indicated for the treatment of lunginfections in patients with cystic fibrosis.

Materials: BisEDT was supplied by Dalton Pharma Services; Lot#ED268-I-II-O I and stored at room temperature. The vehicle used int hisstudy was 0.5% TWEEN 80®, 10 mM sodium phosphate, pH 7.4, in NaCl(adjusted to approximately 300 mOsm) in water which was stored at roomtemperature.

Canines: Male and female canine, beagle dogs,aged 5-7 months were usedint his study. Males weighed 6-10 kg and females weighed 5-9 kg. 6 maleand 6 female were used.

Housing: Animals are housed in indoor or outdoor dog kennels.

Conditioning: Animals are conditioned to restraint and face masks.

Feed: 2025C Harlan Global 25% Protein Diet (Harlan Teklad, Madison,Wis.) once daily (except during conditioning and inhalation exposures).All dogs are fasted the evening prior to all blood samples collected forhematology, clinical chemistry, and coagulation analyses. Each batch offeed is analyzed for contaminants by the manufacturer and used withinthe manufacturer's designated shelf-life. No contaminants are expectedto be present at levels that would interfere with the outcome of thestudy. Municipal water with unlimited access was used, except duringconditioning and inhalation exposures.

Environmental Conditions: The targeted conditions for kennel temperatureand photoperiod will be as follows: Temperature: 18-29° C.; Light Cycle:12-h (on each day of exposure the light cycle may be extended toaccommodate blood sample collections).

Morbidity and Mortality: Animals are observed twice daily for morbidityand mortality. If found moribund, animals will be euthanized by anoverdose of an approved euthanasia solution. Only healthy animals arestudied.

Once adequately conditioned to the restraint device and face-masks, theanimals are individually exposed to the test article aerosols. Theexperimental design is shown in Table 17. Animals receive a singleface-mask inhalation exposure in a ramp study design to test articleaerosols for up to 60 min. Animal observations during and post exposuredetermine the tolerance of inhaled BisEDT aerosols.

Group 1 (vehicle) are exposed to a vehicle atmosphere for 30 minutes.Group 2 (50 μg/kg) are exposed to BisEDT for 30 minutes. Targetconcentrations and exposure durations for Groups 3-5 are based on thepresence of any toxicity via clinical observations or gross necropsyfindings. In each of Groups 3-5, if no adverse clinical signs oftoxicity are observed, the next groups' exposure will be conducted at ahigher target dose. If adverse clinical signs of toxicity are observed,the next groups' exposure will be conducted at a lower target dose. Thisapproach is repeated for all BisEDT doses. The final documented exposureduration is the maximum tolerated dose or the maximum feasible dose.

Blood is collected for pharmacokinetic (PK) analysis, hematology,clinical chemistry, and coagulation analysis.

At each scheduled timepoint (or in cases of morbidity) animals will beeuthanized.

TABLE 17 Experimental Design Blood Group Exposure^(a) Target DoseGender/Animal IDs Collection^(d,e) 1 Day 0 and Day 7: n/a Male: 1001 NATA Vehicle Female: 1002 2 Day 0 and Day 7: Low Male: 2001 4 hrs (±5min), Day BisEDT 50 μg/kg Female: 2002 1, Day 2, Day 7 3 Day 0 and Day7: Low-Mid: Male: 3001 (pre-exposure), BisEDT TBD^(c) Female: 3002 Day 7(post- 4 Day 0 and Day 7: Mid-High: Male: 4001, 4003 exposure), Day 8,BisEDT TBD^(c) Female: 4002, 4004 Day 9, Day 10, 5^(b) Day 0 and Day 7:High: Male: 5001 Day 11, Day 12, BisEDT TBD^(c) Female: 5002 Day 13, Day14 ^(a)All groups consist of 1 male and 1 female animal exposed on Day 0and Day 7. All Groups are terminal on Day 14. Group 4 includes 2additional animals (1 female and 1 male) with necropsy performed on Day21. ^(b)Group 5 will be included if Group 4 does not show signs oftoxicity. ^(c)TBD: Concentrations will be documented by memo andincluded in the final study report and will be based on the results ofthe previous exposure group. ^(d)Blood collections for the additionalanimals (4003 and 4004) in Group 4 are: Day 15, Day 16, Day 17, Day 18,Day 19, Day 20, Day 21. ^(e)Blood collection for PK analysis.

BisEDT formulation: The initial 5.0 mg/ml BisEDT formulation is preparedin 0.5% TWEEN 80®, 10 mM sodium phosphate, pH 7.4, in NaCl (adjusted toapproximately 300 mOsm). On each exposure day the formulation isanalyzed.

Test Article Administration: A representative schematic is presented inFIG. 41 . The aerosol generation system couples a commercially availablecompressed air jet nebulizer to a chamber that allows the aerosols totransition to the animals. Exposure is by face-mask inhalation per thestudy design above. Exposure durations do not exceed 60 min. Formulationconcentration and exposure duration are modulated to achieve targetdoses if necessary.

Animals will be transported to the exposure room and placed ontoexposure tables with restraint harnesses. Face-masks connected to thechamber will be placed on the animal immediately prior to the beginningof the exposure period. Temperature and exposure chamber oxygen (%) willbe monitored throughout the exposure.

Concentration Monitoring: Aerosol concentration monitoring is conductedby collecting aerosols onto pre-weighed GF/A 47-mm filters. The filtersare sampled from the exposure chamber throughout the exposure. Theaerosol sampling flow rate through GF/A filters is maintained at 0.5±0.1L/min. After sample collection, filters are weighed to determine thetotal aerosol concentration in the exposure system. The filters areextracted and analyzed. Based on BisEDT average exposure aerosolconcentration the deposited dose is calculated.

Particle Size Determination: Particle size distribution of aerosols aremeasured from the breathing zone of the exposure chamber by aMercer-style, seven-stage cascade impactor (Intox Products, Inc.,Albuquerque, NM). The particle size distribution is determined in termsof mass median aerodynamic diameter (MMAD) and geometric standarddeviation (GSD). Cascade impactor sample is collected at a flow rate of2.0±0.1 L/min.

Determination of Pulmonary Dose: Deposited dose is calculated using theequation below. In this calculation, the average aerosol concentrationmeasured from the exposure along with the individual animals' bodyweight for each specific exposure day will be used. In this manner, theestimated amount of BisEDT that is deposited in the lungs will becalculated using the measured BisEDT aerosol concentration.

${{DD}\left( {{µg}/{kg}} \right)} = \frac{{{AC}\left( {{µg}/L} \right)} \times {{RMV}\left( {L/{\min.}} \right)} \times {DF} \times {T\left( {\min.} \right)}}{{BW}({kg})}$

-   Where:-   Deposited Dose=(DD) μg/kg-   Respiratory minute volume (RMV)=0.608×BW^(0.852) (Alexander D J et    al., 2008)-   Aerosol exposure concentration (AC)=BisEDT aerosol concentration    (μg/L)-   Deposition Fraction (DF)=assumed deposition fraction of 25%-   BW=body weight of the individual animal on study (kg)

Observations and Measurements

Clinical Observations and Mortality/Morbidity: Detailed clinicalobservations are recorded starting on dose day with observationsrecorded twice per day (morning and afternoon) from arrival to the dayof exposure. General observations include but are not limited to apnea,labored breathing, malaise, marked nasal discharge, etc. Specialattention are paid to clinical signs associated with the respiratorytract. Animals showing severe signs of distress are euthanized at thediscretion of the Study Director and in consultation with veterinarystaff . Examinations are oriented toward (1) identifying dead, weak, ormoribund animals, and (2) documenting the onset and progression of anyabnormal clinical signs. Moribund or dead animals are necropsied as soonas possible after being found but in no case later than 16 hrs afterbeing found.

Body Weights: All animals are weighed after release from quarantine andthat weight will be the pre-study body weight used to randomize animalsinto dose groups. Body weights are collected in the morning prior toexposure and at necropsy.

Blood Collections and Bioanalytical Analysis: Blood are collected forpharmacokinetic (PK) analysis prior to exposure, then at 4 hr (±5 min)post exposure, and again on Day 1, Day 2, Day 7 (pre- and 4 hr (±5min)-post exposure), Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, andDay 14. A total of 0.25 mL is collected at each timepoint. No PK bloodis collected from the vehicle control (Group 1) animals; only animalsexposed to BisEDT have blood collected for PK analysis. Collectedsamples are flash frozen without processing for shipment to Medpace forbioanalytical analysis. Additional blood is collected from all animalsin all groups for hematology (1 mL) and clinical chemistry (1 mL) atbaseline, Day 7 (pre-exposure), and again at euthanasia. Blood (2-2 mL)is collected from all animals at necropsy for coagulation analysis(d-Dimer, fibrinogen, PT, and PTT).

Blood samples are collected from either the jugular vein or anotherperipheral vein (cephalic or saphenous).

Blood for pharmacokinetic analysis (0.25 mL) is collected into tubescontaining K3EDTA as an anticoagulant. The tubes are flash frozen withliquid nitrogen and stored without processing at −70 to −90° C. untilshipping for analysis using ICP-MS assay for quantitation of bismuth asa surrogate for BisEDT.

For hematology analyses, whole blood (1 ml) will be collected withvacutainers containing K₃EDTA as an anticoagulant.

For clinical chemistry analyses, whole blood (1 mL) is placed into serumseparator or clot tube and processed to plasma by centrifugation at aminimum of 1300 g at 2 to 8° C. for 10 (±1) min. Plasma samples arestored at −70 to −90° C. until analysis.

For coagulation analyses, blood (2-2 mL samples) are collected intotubes containing sodium citrate anticoagulant, processed, and thecitrated plasma frozen and held for analysis. Prothrombin Time (PT),activated Partial Thromboplastin Time (aPTT), d-Dimer, and fibrinogenare analyzed on each animal. Samples will be centrifuged (1500 rpm, 15minutes), plasma aliquoted into cryogenic vials, and stored frozen (−70to −90° C.) until analysis.

Hematology and Clinical Chemistry: All dogs are fasted the evening priorto all blood samples collected for hematology, clinical chemistry, andcoagulation analyses.

Hematology samples are analyzed by automated (ADVIA™ 120 HematologySystem, Siemens Medical Solutions Diagnostics, Tarrytown, N.Y.)analyses. Disposition will be recorded on the sample processing form.Parameters for hematology are shown in Table 18.

Clinical chemistry samples are analyzed on a Hitachi Modular AnalyticsClinical Chemistry System (Roche Diagnostics, Indianapolis, Ind.). Theclinical chemistry parameters are shown in Table 19.

If target collection volumes needed for hematology or clinical chemistryanalysis are not obtained, no analyses will be performed for thatanimal. In addition, if evaluations cannot be performed, a reason andnotation will be included in the study file.

TABLE 18 Hematology Parameters Parameter Abbreviation^(a) Units RedBlood Cell Count RBC 10⁶/μL Hemoglobin HGB g/dL Hematocrit HCT % MeanCorpuscular Volume MCV fL Mean Corpuscular Hemoglobin Concentration MCHCg/dL Mean Corpuscular Hemoglobin MCH pg Platelet Count PLT 10³/μLPercent Reticulocytes RETIC % RBC White Blood Cell Count and AbsoluteDifferential White Blood Cell Count WBC 10³/μL Neutrophils PMN 10³/μLLymphocytes LYM 10³/μL Monocytes MONO 10³/μL Eosinophils EOS 10³/μLBasophils BASO 10³/μL Large Unstained Cells LUC 10³/μL ^(a)Abbreviationsfrom the hematology system differ from those listed above, however thefinal results will be reported as described above

TABLE 19 Clinical Chemistry Parameters Analyte Abbreviation^(a) UnitsAlanine Aminotransferase (Alanine ALT IU/L Transaminase)-Serum AlbuminALB g/dL Aspartate Aminotransferase (Aspartate AST IU/LTransaminase)-Serum Bilirubin (Total) BILI-T mg/dL Blood Urea NitrogenBUN mg/dl Calcium CA mg/dL Chloride (Serum) CL-S mmol/L Cholesterol(Total) CHOL mg/dL Creatinine (Serum) CRE-S mg/dL Glucose GLU mg/dLGamma Glutamyltransferase GGT IU/L Alkaline Phosphatase ALP IU/LPhosphate PHOS mg/dL Potassium (Serum) K-S mmol/L Protein (Total) TPg/dL Sodium (Serum) NA-S mmol/L Triglycerides TRIG mg/dL CalculatedParameters and Ratios Albumin/Globulin A/G None Blood UreaNitrogen/Creatinine BUN/CRE None Globulin GLOBN g/dL ^(a)Abbreviationsfrom the chemistry system differ from those listed above, however thefinal results will be reported as described above

Euthanasia, Necropsy, and Histology

Euthanasia: At scheduled necropsies (Day 14 or Day 21, respectively) orin cases of moribundity, animals are tranquilized and euthanized by aveterinarian or their. Animals are tranquilized by administration ofacepromazine (0.02-0.2 mg/kg, IM) and butorphanol (≥0.33 mg/kg, IM).After sedation, an intravenous catheter is placed to accommodateadministration of a ketamine/diazepam cocktail and flushed with a salinesolution. The cocktail is a proportional mixture of 1 mL of ketamine(100 mg/mL) and 1 mL of diazepam (5 mg/mL). The cocktail is then beadministered at a dose of ≥1 mL/9 kg body weight. Note: If a dog issufficiently sedated after use of acepromazine and butorphanol,administration of ketamine/diazepam cocktail may not be required oradministered and will be documented. The animal is then euthanized by anoverdose of a barbiturate-based sedative (EUTHASOL®, ≥1 mL/4.5 kg, IV)and flushed with a saline solution. If needed, exsanguination may beperformed.

Necropsy: Detailed gross necropsies are performed on all animals andwill consist of a complete external and internal examination includingbody orifices (ears, nostrils, mouth, anus, etc.) and cranial, thoracic,and abdominal organs and tissues. All gross findings are recorded indescriptive terms, typically including location(s), size (in mm), shape,color, consistency, and number. Animals found dead will be refrigerateduntil necropsy can be performed. A cause of death is determined ifpossible.

The left lobe of the lung is used for histopathology and will be fixedin 10% NBF. The right middle lobe has three approximately 1 gramsections collected and flash frozen with liquid nitrogen forbioanalytical analysis; samples will be stored at −70 to −90° C. untilshipping for analysis using ICP-MS assay for quantitation of bismuth asa surrogate for BisEDT. Liver, spleen, kidney, brain, heart, andrespiratory tract are collected and fixed for potential, futureanalysis.

Histology and Pathology: Lungs are, prepared, and embedded, cut andmounted on slides, stained with hematoxylin and eosin, and evaluated bya Pathologist. In addition, representative lesions observed andcollected during necropsy may be evaluated. Histology is conducted onGroup 1 and Group 4; additional analyses may be conducted by Amendmentto the Approved Study Protocol.

Results

Table 20 shows the actual deposited dose of BisEDT in the lung for dogstested. Table 21 shows hematology results and Table 22 shows clinicalchemistry results for the dogs tested.

TABLE 20 Actual lung deposited doses per group/animal #: Lung depositedAnimal # Group dose, ug/kg 1001 1 0 1002 1 0 2001 2 55 2002 2 56 3001 3112 3002 3 111 4001 4 176 4002 4 180 4003 4 195 4004 4 197

TABLE 21 Hematology Summary RET Subject/QC Sex/QC WBC RBC HGB HCT MCVMCH MCHC PLT NEUT LYMP MONO EOS BASO LUC % Lot Identifier {circumflexover ( )}3/uL {circumflex over ( )}6/uL g/dL % fL pg g/dL {circumflexover ( )}3/uL {circumflex over ( )}3/uL {circumflex over ( )}3/uL{circumflex over ( )}3/uL {circumflex over ( )}3/uL {circumflex over( )}3/uL {circumflex over ( )}3/uL % 1001 Male 13.48 6.75 15.9 46.6 69.123.5 34.0 250 8.56 3.73 0.82 0.15 0.20 0.03 0.72 1001 Male 7.98 5.8913.8 39.8 67.7 23.4 34.7 241 5.00 2.26 0.54 0.08 0.07 0.02 0.34 1001Male 9.32 6.15 14.3 42.8 69.6 23.2 33.4 106 6.37 2.22 0.57 0.05 0.080.01 0.39 1002 Female 9.18 7.54 17.0 51.2 67.9 22.6 33.2 297 3.97 3.890.39 0.73 0.17 0.02 0.51 1002 Female 8.67 6.75 16.0 44.6 66.1 23.6 35.8168 5.92 1.83 0.68 0.10 0.13 0.01 0.23 1002 Female 7.98 6.47 15.0 45.269.9 23.3 33.3 193 5.38 1.75 0.72 0.07 0.06 0.02 0.44 Grp 1 Avg 11.337.15 16.5 48.9 68.5 23.1 33.6 273.5 6.27 3.81 0.61 0.44 0.19 0.03 0.62Grp 1 Avg 8.33 6.32 14.9 42.2 66.9 23.5 35.3 204.5 5.46 2.05 0.61 0.090.10 0.02 0.29 Grp 1 Avg 8.65 6.31 14.7 44.0 69.8 23.3 33.4 149.5 5.881.99 0.65 0.06 0.07 0.02 0.42 2001 Male 9.18 6.83 15.6 45.8 67.0 22.834.1 197 5.31 2.81 0.63 0.30 0.12 0.01 0.34 2001 Male 9.13 6.49 15.043.1 66.4 23.2 34.9 166 5.11 2.73 0.67 0.45 0.15 0.01 0.55 2001 Male5.99 6.28 14.2 41.8 66.5 22.7 34.1 161 2.88 2.09 0.53 0.39 0.08 0.020.60 2002 Female 6.99 7.05 16.4 49.0 69.5 23.2 33.4 206 3.96 2.27 0.500.14 0.11 0.02 0.65 2002 Female 7.77 6.74 15.0 43.7 64.8 22.3 34.4 2944.26 2.82 0.51 0.09 0.10 0.01 0.25 2002 Female 8.10 6.50 14.3 42.9 66.021.9 33.2 304 4.83 2.61 0.51 0.06 0.07 0.01 0.31 Grp 2 Avg 8.09 6.9416.0 47.4 68.3 23.0 33.8 201.5 4.64 2.54 0.57 0.22 0.12 0.02 0.50 Grp 2Avg 8.45 6.62 15.0 43.4 65.6 22.8 34.7 230.0 4.69 2.78 0.59 0.27 0.130.01 0.40 Grp 2 Avg 7.05 6.39 14.3 42.4 66.3 22.3 33.7 232.5 3.86 2.350.52 0.23 0.08 0.02 0.46 3001 Male 8.74 6.11 13.6 40.2 65.8 22.3 33.9310 4.36 3.29 0.44 0.57 0.07 0.02 0.61 3001 Male 7.25 6.35 14.2 41.064.6 22.3 34.5 321 3.52 2.93 0.45 0.27 0.07 0.02 0.79 3001 Male 12.976.98 15.8 48.2 69.1 22.7 32.8 255 7.28 4.35 0.83 0.38 0.11 0.02 1.023002 Female 11.50 7.17 16.2 47.3 65.9 22.6 34.3 325 6.77 3.67 0.67 0.170.20 0.03 0.91 3002 Female 9.18 6.12 14.6 43.1 70.4 23.9 34.0 247 5.292.86 0.79 0.12 0.10 0.02 0.94 3002 Female 14.08 7.36 16.7 49.8 67.6 22.733.5 319 9.25 3.80 0.76 0.15 0.10 0.02 0.72 Grp 3 Avg 10.12 6.64 14.943.8 65.9 22.5 34.1 317.5 5.57 3.48 0.56 0.37 0.14 0.03 0.76 Grp 3 Avg8.22 6.24 14.4 42.1 67.5 23.1 34.3 284.0 4.41 2.90 0.62 0.20 0.09 0.020.87 Grp 3 Avg 13.53 7.17 16.3 49.0 68.4 22.7 33.2 287.0 8.27 4.08 0.800.27 0.11 0.02 0.87 4001 Male 8.23 6.44 14.8 42.7 66.3 23.0 34.7 2084.61 2.68 0.39 0.48 0.07 0.01 0.41 4001 Male 10.09 6.81 16.0 47.5 69.723.4 33.6 214 5.96 3.18 0.37 0.50 0.07 0.02 0.33 4001 Male 9.86 6.3714.6 45.4 71.3 22.9 32.2 213 6.30 2.78 0.37 0.35 0.04 0.01 0.58 4004Female 10.41 6.46 14.7 41.9 64.8 22.8 35.2 246 5.98 2.88 0.49 0.95 0.090.02 0.27 4004 Female 10.37 7.19 16.6 50.6 70.4 23.1 32.8 238 5.95 3.040.50 0.77 0.09 0.01 0.62 4004 Female 10.50 6.71 15.4 47.6 70.9 22.9 32.4251 6.74 2.82 0.39 0.47 0.06 0.01 0.43 Grp 4 Avg 9.32 6.45 14.75 42.3065.55 22.90 34.95 227.00 5.30 2.78 0.44 0.72 0.08 0.02 0.34 Grp 4 Avg10.23 7.00 16.30 49.05 70.05 23.25 33.20 226.00 5.96 3.11 0.44 0.64 0.080.02 0.48 Grp 4 Avg 10.18 6.54 15.00 46.50 71.10 22.90 32.30 232.00 6.522.80 0.38 0.41 0.05 0.01 0.51 4002 Female 10.21 5.68 13.7 39.8 70.1 24.134.4 257 6.73 2.46 0.73 0.18 0.09 0.01 1.07 4002 Female 7.45 6.61 13.840.7 61.6 20.9 34.0 308 4.11 2.59 0.50 0.13 0.10 0.02 0.88 4003 Male6.63 6.11 12.8 37.1 60.8 21.0 34.6 313 3.60 2.32 0.45 0.19 0.04 0.020.51 4003 Male 11.18 5.92 14.3 44.2 74.7 24.2 32.4 248 7.39 2.87 0.610.21 0.10 0.01 0.83 Grp 4 Avg 8.42 5.90 13.25 38.45 65.45 22.55 34.50285.00 5.17 2.39 0.59 0.19 0.07 0.02 0.79 Grp 4 Avg 9.32 6.27 14.0542.45 68.15 22.55 33.20 278.00 5.75 2.73 0.56 0.17 0.10 0.02 0.86

TABLE 22 Clinical Summary Na K Cl GLUC BUN CREA P TBIL ALPK ALT AST GGTTP ALB CA2 TRIG CHOL GLOB Subject/QC Lot Sex/QC Identifier mmo/L mmo/Lmmo/L mg/dL mg/dL mg/dL mg/dL mg/dL IU/L IU/L IU/L IU/L g/dL g/dL mg/dLmg/dL mg/dL g/dL AGR 1001 Male 149 4.7 109 90 14 0.7 7.6 0.15 248 25 293 5.0 3.3 11.0 53 132 1.7 1.9 1001 Male 146 4.7 111 78 15 0.5 6.4 0.15243 29 29 3 5.0 3.3 10.6 39 133 1.7 1.9 1001 Male 150 4.2 113 92 17 0.56.6 0.15 235 21 33 3 5.5 3.7 10.9 38 130 1.8 2.1 1002 Female 148 5.1 10992 13 0.7 6.4 0.15 91 24 27 3 4.9 3.3 10.3 37 107 1.6 2.1 1002 Female147 4.9 113 88 15 0.7 6.4 0.15 125 26 33 3 5.0 3.6 10.7 32 129 1.4 2.61002 Female 148 4.5 113 89 13 0.7 6.2 0.15 120 27 34 3 5.0 3.5 10.4 29116 1.5 2.3 Grp 1 Avg 149 4.9 109 91 14 0.7 7.0 0.15 170 25 28 3 5.0 3.310.7 45 120 1.7 2.0 Grp 1 Avg 147 4.8 112 83 15 0.6 6.4 0.15 184 28 31 35.0 3.5 10.7 36 131 1.6 2.3 Grp 1 Avg 149 4.4 113 91 15 0.6 6.4 0.15 17824 34 3 5.3 3.6 10.7 34 123 1.7 2.2 2001 Male 149 4.5 109 91 12 0.7 6.80.15 130 22 48 3 5.0 3.3 10.9 66 168 1.7 1.9 2001 Male 147 4.9 113 97 160.6 5.9 0.15 140 22 36 3 5.1 3.4 10.8 80 167 1.7 2.0 2001 Male 147 4.7108 102 15 0.6 5.5 0.15 128 22 36 3 5.5 3.5 11.0 63 152 2.0 1.8 2002Female 149 5.0 110 95 13 0.6 7.4 0.15 241 18 35 3 5.2 3.3 10.9 38 1381.9 1.7 2002 Female 147 5.2 114 98 14 0.6 6.4 0.15 111 29 41 3 5.2 3.610.8 39 123 1.6 2.3 2002 Female 148 4.6 113 89 14 0.6 5.7 0.15 103 27 383 5.3 3.7 10.6 31 114 1.6 2.3 Grp 2 Avg 149 4.8 110 93 13 0.7 7.1 0.15186 20 42 3 5.1 3.3 10.9 52 153 1.8 1.8 Grp 2 Avg 147 5.1 114 98 15 0.66.2 0.15 126 26 39 3 5.2 3.5 10.8 60 145 1.7 2.2 Grp 2 Avg 148 4.7 11196 15 0.6 5.6 0.15 116 25 37 3 5.4 3.6 10.8 47 133 1.8 2.1 3001 Male 1464.7 111 100 13 0.6 6.5 0.15 142 26 30 3 4.8 3.2 10.7 26 100 1.6 2.0 3001Male 146 5.1 110 88 17 0.7 6.8 0.15 135 24 32 3 5.1 3.4 10.5 34 111 1.72.0 3001 Male 148 5.8 109 51 33 1.0 7.2 0.15 121 22 33 3 5.0 3.4 10.0 52111 1.6 2.1 3002 Female 149 5.5 112 112 11 0.6 6.6 0.15 164 28 30 3 5.03.5 11.2 34 126 1.5 2.3 3002 Female 145 5.3 109 90 12 0.5 6.5 0.15 90 2028 3 5.5 3.5 10.8 43 175 2.0 1.8 3002 Female 149 5.9 110 65 25 0.9 7.50.15 176 20 30 3 5.1 3.7 10.4 58 141 1.4 2.6 Grp 3 Avg 148 5.1 112 10612 0.6 6.6 0.15 153 27 30 3 4.9 3.4 11.0 30 113 1.6 2.2 Grp 3 Avg 1465.2 110 89 15 0.6 6.7 0.15 113 22 30 3 5.3 3.5 10.7 39 143 1.9 1.9 Grp 3Avg 149 5.9 110 58 29 1.0 7.4 0.15 149 21 32 3 5.1 3.6 10.2 55 126 1.52.4 4001 Male 152 4.7 120 102 11 0.7 6.5 0.15 103 27 32 3 5.2 3.6 10.849 153 1.6 2.3 4001 Male 147 4.8 116 82 12 0.7 6.6 0.15 114 27 39 3 5.33.4 10.6 46 147 1.9 1.8 4001 Male 145 4.7 113 86 11 0.6 6.0 0.15 119 2834 3 4.9 3.2 10.4 40 111 1.7 1.9 4004 Female 153 4.6 119 102 13 0.6 5.90.15 103 28 33 3 5.1 3.4 10.8 53 125 1.7 2.0 4004 Female 148 5.1 115 7013 0.6 6.3 0.15 93 25 38 3 5.2 3.3 10.8 49 124 1.9 1.7 4004 Female 1464.3 114 96 13 0.6 5.6 0.15 95 24 31 3 5.0 3.3 10.6 44 112 1.7 1.9 Grp 4Avg 153 4.7 120 102 12 0.7 6.2 0.15 103 28 33 3 5.2 3.5 10.8 51 139 1.72.2 Grp 4 Avg 148 5.0 116 76 13 0.7 6.5 0.15 104 26 39 3 5.3 3.4 10.7 48136 1.9 1.8 Grp 4 Avg 146 4.5 114 91 12 0.6 5.8 0.15 107 26 33 3 5.0 3.310.5 42 112 1.7 1.9 4002 Female 153 5.3 116 106 13 0.6 6.4 0.15 147 2032 3 5.6 3.5 11.1 34 182 2.1 1.7 4002 Female 149 5.3 112 79 12 0.5 6.90.15 103 21 35 3 5.5 3.5 11.2 41 169 2.0 1.8 4003 Male 149 4.9 117 10916 0.6 7.2 0.15 146 40 30 3 5.4 3.5 11.2 55 152 1.9 1.8 4003 Male 1474.9 112 81 15 0.7 7.1 0.15 129 23 33 3 5.4 3.3 11.0 53 176 2.1 1.6

REFERENCES

Alexander D J, Collins C J, Coombs D W, Gilkison, I S, Hardy, C J,Healey, G, Karantabias, G, Johnson, N, Karlsson, A, Kilgour, J D, andMcDonald, P. Association of Inhalation Toxicologists (AIT) working partyrecommendation for standard delivered dose calculation and expression innon-clinical aerosol inhalation toxicology studies with pharmaceuticals.Inhalation Toxicology; 20(13): 1179-89, 2008.

National Research Council, 2011. Guide for the Care and Use ofLaboratory Animals. National Academy Press, Washington, DC.

Tepper, J S, Kuehl, P K, Cracknell, S, Nikula, K J, Pei, L, andBlanchard, J D. 2016. Symposium Summary: “Breath In, Breath Out, ItsEasy: What You Need to Know About Developing Inhaled Drugs.” Int. J.Toxicol. 35(4) 376-392.

Example 10 Antimicrobial Interaction of BisEDT with Agents Used to TreatCystic Fibrosis Infections Caused by Pseudomonas aeruginosa andBurkholderia cepacia Complex

Introduction: The interaction between BisEDT and a variety of agentsused in the treatment of patients with cystic fibrosis (CF) wasevaluated against P. aeruginosa and Burkholderia spp. The antimicrobialinteraction was determined by measuring fractional inhibitoryconcentrations (FIC) in a checkerboard assay.

Materials and Methods

Test articles: BisEDT was provided as a dry powder and was stored atroom temperature in the dark prior to testing. The comparator compoundswere handled in accordance with guidelines from the Clinical andLaboratory Standards Institute (CLSI; 1, 2). Specific information on theindividual drug lots and concentration ranges tested is shown in theTable 23 below:

TABLE 23 drug lots and concentration ranges tested ConcentrationConcentration Ranges tested Ranges tested Test Articles Supplier LotNumber Solvent (μg/ml) (MIC) (μg/ml) (FIC) BisEDT MicrobionED268-1-11-01 DMSO 64-0.06 16-0.25; 4-0.06 Tobramycin Sigma 109K1184Sterile dH₂O 256-0.25; 2-0.002 256-0.25 Amikacin Sigma 058K0803 SteriledH₂O 256-0.25; 2-0.002 256-0.25 Aztreonam USP R041F0 Saturated 256-0.25;2-0.002 256-0.25 NaHCO₃ Meropenem USP J0K434 Sterile dH₂O 256-0.25;2-0.002  64-0.06 Ciprofloxacin USP R05170 Sterile dH₂O 256-0.25; 2-0.002256-0.25 Colistin Sigma SLBV1747 Sterile dH₂O 256-0.25; 2-0.002 256-0.25Appropriate solvents were added to the drugs which were prepared at40-fold the top testing concentration. The stock solutions were allowedto stand for approximately 1 hr at room temperature in the dark toauto-sterilize before being used for testing. Drug stocks of thecomparators were frozen and stored at −80° C.

Organisms: The test organisms were clinical isolates previously acquiredby Micromyx or from the American Type Culture Collection (ATCC). Uponreceipt at Micromyx, the isolates were streaked under suitableconditions onto agar medium appropriate to each organism. The organismswere incubated for 18-24 hr at 35° C. Colonies harvested from thesegrowth plates were resuspended in the appropriate medium containing acryoprotectant. Aliquots of each suspension were then frozen at −80° C.Prior to the assay, the organisms were streaked onto trypticase soy agarplus 5% sheep blood (BD; Sparks, Md.; Lot No. 8179506) and wereincubated as described above.

Test Media: The medium employed for the assay was cation-adjustedMueller Hinton broth (MHB II; Becton-Dickinson, Sparks, Md.; Lot No8096574). The medium was prepared according to CLSI guidelines (2).

MIC Assay Methodology: MIC assay plates were prepared using the CLSIbroth microdilution procedure (1, 2). Automated liquid handlers(Multidrop 384, Biomek 2000 and Biomek FX) were used to conduct serialdilutions and liquid transfers. All wells in columns 2 through 12 of astandard 96-well microdilution plate (Costar 3795) were filled with 150μL of the proper diluent. Three hundred μL of each test drug (at 40×)were added to each well in Column 1 of the plates. This plate was usedto prepare the drug “mother plate” which provided the serial drugdilutions for the replicate “daughter plates”. The Biomek 2000 was usedto complete the serial transfers through Column 11 in the mother plates.The wells of Column 12 contained no drug and were the organism growthcontrol wells.

The daughter plates were loaded with 185 μL per well of the CAMHBdescribed using the Multidrop 384. The daughter plates were completed onthe Biomek FX instrument which transferred 5 μL of drug solution fromeach well of a mother plate to each corresponding well of each daughterplate in a single step.

A standardized inoculum of each organism was prepared per CLSI methods(2). Suspensions were prepared to equal a 0.5 McFarland standard,followed by dilution in test media 1:20. The inocula were dispensed intosterile reservoirs divided by length (Beckman Coulter) and the Biomek2000 was used to inoculate the plates. Daughter plates were placed onthe Biomek 2000 work surface in reverse orientation so that inoculationtook place from low to high drug concentration. The Biomek 2000delivered 10 μL of the diluted suspension into each well resulting in afinal concentration of approximately 5×10⁵ CFU/mL. Plates were stacked3-4 high, covered with a sterile lid on the top plate, placed in plasticbags, and incubated at 35° C. for approximately 18 hr.

The microplates were viewed from the bottom using a plate viewer and theMIC was read. The MIC was recorded as the lowest concentration of drugthat inhibited visible growth of the organism. Uninoculated solubilitycontrol plates were also observed for evidence of drug precipitation.

FIC Assay Procedure: FIC test ranges were set based on brothmicrodilution MIC test data. FIC assay plates were prepared using theCLSI broth microdilution procedure (1, 2) and automated liquid handlers(Multidrop 384, Biomek 2000 and Biomek FX) to conduct serial dilutionsand liquid transfers.

The wells of a standard 96-well microdilution plate (Costar) were filledwith 150 μL of the appropriate diluent in columns 2 through 12. A 300 μLaliquot at 40× the highest final concentration to be tested was added toeach well in Column 1 of the plate. The Biomek 2000 was used to makeeleven 2-fold serial dilutions in the “combination agent mother” platefrom columns 2 through 11.

The wells of the “test agent mother” plate were filled with 150 μL ofdiluent in rows B-H. Row A of this plate was filled with 300 μL of thetest agent stock solutions at 40× the highest final concentration to betested. Serial 2-fold dilutions were then prepared from row B-G by handusing a multichannel pipette.

The “daughter plates” were loaded with 180 μL of cation-adjustedMueller-Hinton broth (CAMHB) using the Multidrop 384. The Biomek FX wasused to transfer 5 μL of drug solution from each well of the combinationagent mother plate to the corresponding well in all of the daughterplates in a single step. Then a 5 μL aliquot from each well of the testagent mother plate was transferred with the Biomek FX into thecorresponding well of the daughter plate. Row H and Column 12 eachcontained serial dilutions of combination agent and the test agentalone, respectively, for determination of the MIC. This procedure wasrepeated for test agents and combination agents evaluated.

A standardized inoculum of each organism was prepared per CLSI methods(2). Colonies were picked from the primary plate and a suspension wasprepared to equal a 0.5 McFarland turbidity standard. The suspensionswere additionally diluted 1:20. A 10 μL standardized inoculum wasdelivered into each well using the Biomek 2000 from low to highconcentration. These inoculations yielded a final cell concentration inthe daughter plates of approximately 5×10⁵ CFU/mL in each well.

The test format resulted in the creation of an 8×12 checkerboard whereeach compound was tested alone (Column 12 and Row H) and in combinationat varying ratios of drug concentration (see FIG. 43 ).

Plates were stacked 3-4 high, covered with a sterile lid on the topplate, placed in plastic bags, and incubated at 35° C. for approximately18 hr (with the exception of P. aeruginosa isolate 8798 which wasincubated for 42 hr due to poor growth at 18 hr). Plates were viewedfrom the bottom using a plate viewer. Prepared reading sheets weremarked for the MIC of the combination agent (row H), the MIC of testagent (column 12), and the wells of the growth-no growth interface forwells containing test agent and combination agent at varying ratios. TheFIC was read and recorded as the lowest concentration of drug thatexhibited no growth of the organism by row where agents were tested incombination (rows B through G). Pinpoint trailing was not interpreted asgrowth.

FIC/FICI Calculations: FICs were calculated essentially as described byEliopoulos, et al. (3), as applicable.

For each relevant row of the panel, the FIC index (FICI) was calculatedas below:

FIC_(drug A)/MIC_(drug A)+FIC_(drug B)/MIC_(drug B)=FIC index (FICI)

Mean FICI were determined for the combination.

In the instance where an MIC for one of the test agents was off-scale(greater than the highest test concentration evaluated, e.g. >32 pg/mL),the MIC was set to the next highest 2-fold concentration fordetermination of the FIC (e.g. if the MIC was >32 μg/mL, the FIC wascalculated based on an MIC of 64 μg/mL).

Using the criteria described by Odds (4), the mean FICI for thecombination was interpreted as follows:≤0.5=synergy, >0.5-4=additive/indifferent, and >4=antagonism.

An interpretation of “synergy” is consistent with inhibition of organismgrowth by combinations at concentrations significantly below (>4-fold)the MIC of either compound alone, resulting in a low FICI value (≤0.50).An interpretation of “indifference” is consistent with growth inhibitionat concentrations at or slightly below/above the MIC values of theindividual compounds alone, resulting in an FICI value of >0.50 but lessthan or equal to 4.0. An interpretation of “antagonism” results when theconcentrations of the compounds in combination that are required toinhibit organism growth are substantially greater (>4-fold) than thosefor the compounds individually, resulting in an FICI value of >4.0.

Results and Discussion

Broth microdilution MIC values for the evaluated agents against the testorganisms as observed during initial MIC testing are summarized in Table24. MIC values for BisEDT and the other test agents against P.aeruginosa ATCC 27853 were within CLSI QC ranges (1). BisEDT maintainedactivity across the evaluated isolates despite the high degree ofresistance to other agents. Based on the resulting phenotypes, isolatesshaded in grey were selected for subsequent evaluation in checkerboardassays with BisEDT in combination with the other test agents.

The MIC values observed during FIC testing are summarized in Table 25.As expected, these results were consistent (typically identical orwithin 2-fold) with those observed during initial MIC testing (Table24). The rare instances where MIC values differed 4-fold with thoseobserved during initial MIC testing are shaded in grey. As duringinitial MIC testing, BisEDT and the other test agents had MIC valueswithin the QC ranges for P. aeruginosa ATCC 27853. The median MIC valueof BisEDT as observed across 6 checkerboard panels is reported in Table25 alongside the MIC range. The MIC values observed with BisEDT wereconsistent across checkerboard panels during FIC testing as expected.

All test data from FIC panels are shown by organism in Tables 28-87. Themean FICI values observed for BisEDT in combination with all evaluatedagents across the selected isolates are summarized in Table 26.Instances where individual FICI values on checkerboard panels exhibitedsynergy/antagonism are also noted.

Excluding colistin and ciprofloxacin, the majority of the interactionsobserved between BisEDT and other agents by FICI wereadditive/indifferent with mean FICI values and individual FICI valuesacross checkerboard panels generally between 0.5 to 4. For a subset ofisolates, synergy between BisEDT and colistin was observed based on meanFICI values ≤0.50; for the lone colistin-R P. aeruginosa (isolate 8798),for both isolates of B. cepacia, and for one isolate of B. cenocepacia(isolate 0548). For one of the P. aeruginosa (isolate 9108), antagonismbetween BisEDT and ciprofloxacin was observed based on a mean FICIvalue >4. For BisEDT in combination with ciprofloxacin, there were 3additional isolates where there was at least one row on the FIC panelthat had an FICI value indicative of antagonism. There was also oneisolate of B. cepacia (isolate 1793) where there was one row with anFICI value indicative of antagonism for BisEDT in combination withmeropenem.

In summary, the overall interaction between BisEDT and other agents usedto treat CF was additive/indifferent against the CF pathogens P.aeruginosa and B. cepacia complex with the exception of select instanceswhere synergy was apparent for BisEDT in combination with colistin andselect instances where antagonism was apparent for BisEDT in combinationwith ciprofloxacin. Whether these instances indicate true synergy orantagonism for these combinations requires further investigation bytime-kill kinetic analysis.

TABLE 24 Summary of activity as observed during initial MIC testing MIC(μg/mL) Organism Isolate ID Phenotype¹ BisEDT MEM TOB AMK CIP AZT COL P.aeruginosa 103 — 1 1 1 2 0.5 8 0.5 (ATCC 25922) (0.5-4)² (0.12-1)(0.25-1) (1-4) (0.12-1) (2-8) (0.5-4) 1497 TOB-R, AMK-R, CIP-I, 0.5 0.0616 64 2 32 4 AZT-R, COL-R 1530 TOB-R, AMK-R, CIP-R 1 0.5 32 64 8 4 0.51553 TOB-R, AMK-R, CIP-R 2 0.5 >256 64 4 8 0.5 6322 MEM-R, TOB-R, AMK-R,2 16 128 64 64 64 0.5 CIP-R, AZT-R 6977 MEM-I, TOB-R, CIP-R, 1 4 128 16128 16 1 AZT-I 7745 MEM-R, CIP-R, AZT-R 2 16 0.5 4 4 32 0.5 7754 MEM-R,TOB-R, CIP-R, 1 32 32 8 8 16 0.5 AZT-I 7762 MEM-I, CIP-I 2 4 1 8 2 8 17871 MEM-R, AZT-I 1 8 0.5 2 0.25 16 0.5 7886 MEM-R, CIP-R, AZT-R 2 32 18 8 64 0.5 7874 — 1 1 0.5 2 0.12 4 1 8797 MEM-R, TOB-R, AMK-R, 0.2532 >256 >256 8 >256 >256 CIP-R, AZT-R, COL-R 8798 MEM-R, TOB-R, AMK-R, 264 32 128 4 256 >256 CIP-R, AZT-R, COL-R 9108 CIP-R, AZT-I 2 0.5 0.5 432 16 0.5

TABLE 25 Summary of activity as observed during initial MIC testing MIC(μg/mL) Organism Isolate ID Phenotype¹ BisEDT MEM TOB AMK CIP AZT COL B.cepacia 0546 — 2 4 64 64 2 16 >256 0547 — 1 2 128 128 1 16 >256 9040 — 14 8 8 1 32 >256 1793 — 0.5 4 64 64 0.5 128 >256 1794 — 2 4 64 128 132 >256 B. cenocepacia 0548 CIP-R 8 4 64 256 8 64 >256 0813 CPI-I 2 2256 >256 4 8 >256 1631 — 2 4 128 256 2 32 >256 1783 — 2 4 128 256 232 >256 8555 — 2 4 128 256 2 256 >256 B. multivorans 1580 CIP-R 2 2 128128 8 64 >256 1791 — 1 4 64 128 1 4 >256 1795 MEM-I, CIP-R 2 8 >256 >25632 4 >256 5665 CIP-I 1 2 64 256 4 4 >256 8952 CIP-I 4 2 16 64 4 8 >256MEM = meropenem, TOB = tobramycin, AMK = amikacin, CIP = ciprofloxacin,AZT = aztreonam, COL = colistin, -R = resistant, -I = intermediate¹Phenotype is based off of MIC interpretation in accordance with CLSIbreakpoints (1) or in the case of colistin and P. aeruginosa EUCASTbreakpoints (v.8.1) Note that B. cepacia complex are intrinsicallyresistant to aminoglycosides, aztreonam, and colistin (1) ² CLSI QCrange shown in parenthesis where applicable Cells shaded grey wereselected for FIC testing

TABLE 26 Summary of activity as observed during FIC testing MIC (μg/mL)Organism Isolate ID Phenotype¹ BisEDT³ MEM TOB AMK CIP AZT COL P.aeruginosa 103 — 1 1 1 4 1 8 0.5 (ATCC 25922)² (0.5-4) (0.12-1) (0.25-1)(1-4) (0.12-1) (2-8) (0.5-4) 6322 MEM-R, TOB-R, AMK-R, 1-2 16 128 64 12832 0.5 CIP-R, AZT-R (2) 6977 MEM-I, TOB-R, CIP-R, 1 8 128 16 128 64 1AZT-I (1) 7745 MEM-R, CIP-R, AZT-R 1-2 16 0.5 4 2 16 0.5 (2) 8798 MEM-R,TOB-R, AMK-R, 2 32 16 128 8 >256 >256 CIP-R, AZT-R, COL-R (2) 9108CIP-R, AZT-I 2-4 1 0.5 4 16 16 0.5 (2) B. cepacia 0546 — 2-4 4 32 64 232 >256 (2, 4) 1793 — 1-2 4 16 32 0.5 128 >256 (1) B. cenocepacia 0548CIP-R 4 4 32 128 2 64 >256 (4) 0813 CIP-I 2-4 2 256 >256 2 32 >256 (2,4) B. multivorans 1795 MEM-I, CIP-R 2 4 >256 >256 32 16 >256 (2) MEM =meropenem, TOB = tobramycin, AMK = amikacin, CIP = ciprofloxacin, AZT =aztreonam, COL = colistin, -R = resistant, -I = intermediate ¹Phenotypedetermined based on initial MIC testing as shown in Table 1 Note that B.cepacia complex are intrinsically resistant to aminoglycosides,aztreonam, and colistin (1) ²CLSI QC range shown in parenthesis for ATCC25922; ATCC 25922 was not tested on checkerboard panels with agents incombination, each agent was tested alone solely for the purpose of QC³With the exception of ATCC 25922 where only one replicate was testedfor the purpose of quality control (QC range shown in parenthesis), theMIC result for BisEDT represents the MIC range and mode as observedacross six different FIC panels.

TABLE 27 Summary of mean FICI data for BisEDT in combination withevaluated agents Isolate Mean FICI ID Phenotype¹ MEM TOB AMK CIP AZT COL6322 MEM-R, TOB-R, 1.19 1.19 2.23 1.19 1.19 1.23 AMK-R, CIP-R, AZT-R6977 MEM-I, TOB-R, 1.73 2.23 2.23 2.23 1.23 1.23 CIP-R, AZT-1 7745MEM-R, CIP-R, 0.64 1.19 1.23 2.59* 0.99 1.19 AZT-R 8798 MEM-R, TOB-R,1.19 0.91 1.09 0.99 0.89 0.22 AMK-R, CIP-R, AZT-R, COL-R 9108 CIP-R,AZT-I 1.19 1.19 1.29 4.50 0.79 1.08 0546 — 1.99 0.69 0.64 1.35 1.16 0.251793 — 2.59* 1.11 1.29 1.98 1.23 0.33 0548 CIP-R 1.11 0.67 0.67 3.23*1.23 0.41 0813 CIP-I 1.08 0.91 0.99 2.85* 1.19 0.72 1795 MEM-I, CIP-R1.33 1.33 1.33 1.19 1.39 1.33 MEM = meropenem, TOB = tobramycin, AMK =amikacin, CIP = ciprofloxacin, AZT = aztreonam, COL = colistin, -R =resistant, -I = intermediate ¹Phenotype determined based on initial MICtesting as shown in Table 1 **indicates that at least one row on thetest panel had an individual FICI value ≤ 0.5 (indicative of synergy)*indicates that at least one row on the test panel had an individualFICI value > 4 (indicative of antagonism)

TABLE 28 MIC (μg/mL), FIC, FICI, for BisEDT in combination withMeropenem Organism: P. aeruginosa 6322 FICI (N): 5 Drug A: BisEDT Drug AMIC: 2 SUM FICI: 5.97 Drug B: Meropenem Drug B MIC: 16 MEAN FICI: 1.19Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 16 1 1.5 D 0.5 0.2516 1 1.25 E 0.25 0.125 16 1 1.125 F 0.12 0.06 16 1 1.06 G 0.06 0.03 16 11.03 H

TABLE 29 MIC (μg/mL), FIC, FICI, for BisEDT in combination withTobramycin Organism: P. aeruginosa 6322 FICI (N): 5 Drug A: BisEDT DrugA MIC: 2 SUM FICI: 5.97 Drug B: Tobramycin Drug B MIC: 128 MEAN FICI:1.19 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 128 1 1.5 D0.5 0.25 128 1 1.25 E 0.25 0.125 128 1 1.125 F 0.12 0.06 128 1 1.06 G0.06 0.03 128 1 1.03 H

TABLE 30 MIC (μg/mL), FIC, FICI, for BisEDT in combination with AmikacinOrganism: P. aeruginosa 6322 FICI (N): 4 Drug A: BisEDT Drug A MIC: 1SUM FICI: 8.93 Drug B: Amikacin Drug B MIC: 64 MEAN FICI: 2.23 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 128 2 2.5 E 0.250.25 128 2 2.25 F 0.12 0.12 128 2 2.12 G 0.06 0.06 128 2 2.06 H

TABLE 31 MIC (μg/mL), FIC, FICI, for BisEDT in combination withCiprofloxacin Organism: P. aeruginosa 6322 FICI (N): 5 Drug A: BisEDTDrug A MIC: 2 SUM FICI: 5.97 Drug B: Ciprofloxacin Drug B MIC: 128 MEANFICI: 1.19 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 128 11.5 D 0.5 0.25 128 1 1.25 E 0.25 0.125 128 1 1.125 F 0.12 0.06 128 11.06 G 0.06 0.03 128 1 1.03 H

TABLE 32 MIC (μg/mL), FIC, FICI, for BisEDT in combination withAztreonam Organism: P. aeruginosa 6322 FICI (N): 5 Drug A: BisEDT Drug AMIC: 2 SUM FICI: 5.97 Drug B: Aztreonam Drug B MIC: 32 MEAN FICI: 1.19Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 16 0.5 1 D 0.5 0.2516 0.5 0.75 E 0.25 0.125 32 1 1.125 F 0.12 0.06 32 1 1.06 G 0.06 0.03 642 2.03 H

TABLE 33 MIC (μg/mL), FIC, FICI, for BisEDT in combination with ColistinOrganism: P. aeruginosa 6322 FICI (N): 4 Drug A: BisEDT Drug A MIC: 1SUM FICI: 4.93 Drug B: Colistin Drug B MIC: 0.5 MEAN FICI: 1.23 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 0.5 1 1.5 E 0.250.25 0.5 1 1.25 F 0.12 0.12 0.5 1 1.12 G 0.06 0.06 0.5 1 1.06 H

TABLE 34 MIC (μg/mL), FIC, FICI, for BisEDT in combination withMeropenem Organism: P. aeruginosa 6977 FICI (N): 4 Drug A: BisEDT Drug AMIC: 1 SUM FICI: 6.93 Drug B: Meropenem Drug B MIC: 8 MEAN FICI: 1.73Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 16 2 2.5 E 0.250.25 16 2 2.25 F 0.12 0.12 8 1 1.12 G 0.06 0.06 8 1 1.06 H

TABLE 35 MIC (μg/mL), FIC, FICI, for BisEDT in combination withTobramycin Organism: P. aeruginosa 6977 FICI (N): 4 Drug A: BisEDT DrugA MIC: 1 SUM FICI: 8.93 Drug B: Tobramycin Drug B MIC: 128 MEAN FICI:2.23 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 256 2 2.5E 0.25 0.25 256 2 2.25 F 0.12 0.12 256 2 2.12 G 0.06 0.06 256 2 2.06 H

TABLE 36 MIC (μg/mL), FIC, FICI, for BisEDT in combination with AmikacinOrganism: P. aeruginosa 6977 FICI (N): 4 Drug A: BisEDT Drug A MIC: 1SUM FICI: 8.93 Drug B: Amikacin Drug B MIC: 16 MEAN FICI: 2.23 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 32 2 2.5 E 0.250.25 32 2 2.25 F 0.12 0.12 32 2 2.12 G 0.06 0.06 32 2 2.06 H

TABLE 37 MIC (μg/mL), FIC, FICI, for BisEDT in combination withCiprofloxacin Organism: P. aeruginosa 6977 FICI (N): 4 Drug A: BisEDTDrug A MIC: 1 SUM FICI: 8.93 Drug B: Ciprofloxacin Drug B MIC: 128 MEANFICI: 2.23 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 2562 2.5 E 0.25 0.25 256 2 2.25 F 0.12 0.12 256 2 2.12 G 0.06 0.06 256 22.06 H

TABLE 38 MIC (μg/mL), FIC, FICI, for BisEDT in combination withAztreonam Organism: P. aeruginosa 6977 FICI (N): 4 Drug A: BisEDT Drug AMIC:  1 SUM FICI: 4.93 Drug B: Aztreonam Drug B MIC: 64 MEAN FICI: 1.23Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 64 1 1.5 E 0.250.25 64 1 1.25 F 0.12 0.12 64 1 1.12 G 0.06 0.06 64 1 1.06 H

TABLE 39 MIC (μg/mL), FTC, FICI, for BisEDT in combination with ColistinOrganism: P. aeruginosa 6977 FICI (N): 4 Drug A: BisEDT Drug A MIC: 1SUM FICI: 4.93 Drug B: Colistin Drug B MIC: 1 MEAN FICI: 1.23 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 1 1 1.5 E 0.25 0.251 1 1.25 F 0.12 0.12 1 1 1.12 G 0.06 0.06 1 1 1.06 H

TABLE 40 MIC (μg/mL), FIC, FICI, for BisEDT in combination withMeropenem Organism: P. aeruginosa 7745 FICI (N): 5 Drug A: BisEDT Drug AMIC: 2 SUM FICI: 3.22 Drug B: Meropenem Drug B MIC: 16 MEAN FICI: 0.64Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 4 0.25 0.75 D 0.50.25 8 0.5 0.75 E 0.25 0.125 8 0.5 0.625 F 0.12 0.06 8 0.5 0.56 G 0.060.03 8 0.5 0.53 H

TABLE 41 MIC (μg/mL), FIC, FICI, for BisEDT in combination withTobramycin Organism: P. aeruginosa 7745 FICI (N): 5 Drug A: BisEDT DrugA MIC: 2 SUM FICI: 5.97 Drug B: Tobramycin Drug B MIC: 0.5 MEAN FICI:1.19 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 0.5 1 1.5 D0.5 0.25 0.5 1 1.25 E 0.25 0.125 0.5 1 1.125 F 0.12 0.06 0.5 1 1.06 G0.06 0.03 0.5 1 1.03 H

TABLE 42 MIC (μg/mL), FIC, FICI, for BisEDT in combination with AmikacinOrganism: P. aeruginosa 7745 FICI (N): 4 Drug A: BisEDT Drug A MIC: 1SUM FICI: 4.93 Drug B: Amikacin Drug B MIC: 4 MEAN FICI: 1.23 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 4 1 1.5 E 0.25 0.254 1 1.25 F 0.12 0.12 4 1 1.12 G 0.06 0.06 4 1 1.06 H

TABLE 43 MIC (μg/mL), FIC, FICI, for BisEDT in combination withCiprofloxacin Organism: P. aeruginosa 7745 FICI (N):  5 Drug A: BisEDTDrug A MIC: 2 SUM FICI: 12.97 Drug B: Ciprofloxacin Drug B MIC: 2 MEANFICI:  2.59 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 4 2 2.5D 0.5 0.25 8 4 4.25 E 0.25 0.125 4 2 2.125 F 0.12 0.06 4 2 2.06 G 0.060.03 4 2 2.03 H

TABLE 44 MIC (μg/mL), FIC, FICI, for BisEDT in combination withAztreonam Organism: P. aeruginosa 7745 FICI (N): 5 Drug A: BisEDT Drug AMIC: 2 SUM FICI: 4.97 Drug B: Aztreonam Drug B MIC: 16 MEAN FICI: 0.99Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 8 0.5 1 D 0.5 0.258 0.5 0.75 E 0.25 0.125 16 1 1.125 F 0.12 0.06 16 1 1.06 G 0.06 0.03 161 1.03 H

TABLE 45 MIC (μg/mL), FIC, FICI, for BisEDT in combination with ColistinOrganism: P. aeruginosa 7745 FICI (N): 5 Drug A: BisEDT Drug A MIC: 2SUM FICI: 5.97 Drug B: Colistin Drug B MIC: 0.5 MEAN FICI: 1.19 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 0.5 1 1.5 D 0.5 0.250.5 1 1.25 E 0.25 0.125 0.5 1 1.125 F 0.12 0.06 0.5 1 1.06 G 0.06 0.030.5 1 1.03 H

TABLE 46 MIC (μg/mL), FIC, FICI, for BisEDT in combination withMeropenem Organism: P. aeruginosa 8798 FICI (N): 5 Drug A: BisEDT Drug AMIC: 2 SUM FICI: 5.97 Drug B: Meropenem Drug B MIC: 32 MEAN FICI: 1.19Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 32 1 1.5 D 0.5 0.2532 1 1.25 E 0.25 0.125 32 1 1.125 F 0.12 0.06 32 1 1.06 G 0.06 0.03 32 11.03 H

TABLE 47 MIC (μg/L), FIC, FICI, for BisEDT in combination withTobramycin Organism: P. aeruginosa 8798 FICI (N): 6 Drug A: BisEDT DrugA MIC:  2 SUM FICI: 5.47 Drug B: Tobramycin Drug B MIC: 16 MEAN FICI:0.91 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5  8 0.5 1 D 0.50.25 16 1 1.25 E 0.25 0.125 16 1 1.125 F 0.12 0.06 16 1 1.06 G 0.06 0.0316 1 1.03 H

TABLE 48 MIC (μg/mL), FIC, FICI, for BisEDT in combination with AmikacinOrganism: P. aeruginosa 8798 FICI (N): 5 Drug A: BisEDT Drug A MIC:  2SUM FICI: 5.47 Drug B: Amikacin Drug B MIC: 128 MEAN FICI: 1.09 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5  64 0.5 1 D 0.5 0.25128 1 1.25 E 0.25 0.125 128 1 1.125 F 0.12 0.06 128 1 1.06 G 0.06 0.03128 1 1.03 H

TABLE 49 MIC (μg/mL), FIC, FICI, for BisEDT in combination withCiprofloxacin Organism: P. aeruginosa 8798 FICI (N): 5 Drug A: BisEDTDrug A MIC: 2 SUM FICI: 4.97 Drug B: Ciprofloxacin Drug B MIC: 8 MEANFICI: 0.99 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 4 0.5 1D 0.5 0.25 4 0.5 0.75 E 0.25 0.125 8 1 1.125 F 0.12 0.06 8 1 1.06 G 0.060.03 8 1 1.03 H

TABLE 50 MIC (μg/mL), FIC, FICI, for BisEDT in combination withAztreonam Organism: P. aeruginosa 8798 FICI (N): 5 Drug A: BisEDT Drug AMIC:    2 SUM FICI: 4.47 Drug B: Aztreonam Drug B MIC: >256 MEAN FICI:0.89 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5   256 0.5 1 D0.5 0.25   256 0.5 0.75 E 0.25 0.125   256 0.5 1.625 F 0.12 0.06 >256 11.06 G 0.06 0.03 >256 1 1.03 H

TABLE 51 MIC (μg/mL), FIC, FICI, for BisEDT in combination with ColistinOrganism: P. aeruginosa 8798 FICI (N): 5 Drug A: BisEDT Drug A MIC:    2SUM FICI: 1.12 Drug B: Colistin Drug B MIC: >256 MEAN FICI: 0.22 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5  8 0.016 0.516 D 0.50.25  8 0.016 0.266 E 0.25 0.125 16 0.031 0.156 F 0.12 0.06 16 0.0310.091 G 0.06 0.03 32 0.063 0.093 H

TABLE 52 MIC (μg/mL), FIC, FICI, for BisEDT in combination withMeropenem Organism: P. aeruginosa 9108 FICI (N): 5 Drug A: BisEDT Drug AMIC: 1 SUM FICI: 5.97 Drug B: Meropenem Drug B MIC: 1 MEAN FICI: 1.19Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 1 1 1.5 D 0.5 0.251 1 1.25 E 0.25 0.125 1 1 1.125 F 0.12 0.06 1 1 1.06 G 0.06 0.03 1 11.03 H

TABLE 53 MIC (μg/mL), FIC, FICI, for BisEDT in combination withTobramycin Organism: P. aeruginosa 9108 FICI (N): 5 Drug A: BisEDT DrugA MIC: 2 SUM FICI: 5.97 Drug B: Tobramycin Drug B MIC: 0.5 MEAN FICI:1.19 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 0.5 1 1.5 D0.5 0.25 0.5 1 1.25 E 0.25 0.125 0.5 1 1.125 F 0.12 0.06 0.5 1 1.06 G0.06 0.03 0.5 1 1.03 H

TABLE 54 MIC (μg/mL), FIC, FICI, for BisEDT in combination with AmikacinOrganism: P. aeruginosa 9108 FICI (N): 5 Drug A: BisEDT Drug A MIC: 2SUM FICI: 4.47 Drug B: Amikacin Drug B MIC: 4 MEAN FICI: 1.29 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 2 0.5 1 D 0.5 0.25 8 22.25 E 0.25 0.125 4 1 1.125 F 0.12 0.06 4 1 1.06 G 0.06 0.03 4 1 1.03 H

TABLE 55 MIC (μg/mL), FIC, FICI, for BisEDT in combination withCiprofloxacin Organism: P. aeruginosa 9108 FICI (N):  6 Drug A: BisEDTDrug A MIC:  4 SUM FICI: 27.00 Drug B: Cipro- Drug B MIC: 16 MEAN FICI: 4.50 floxacin Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B 2 0.5  0.250.01563 0.51563 C 1 0.25 128 8 8.25 D 0.5 0.125 128 8 8.125 E 0.25 0.06364 4 4.063 F 0.12 0.03 64 4 4.03 G 0.06 0.015 32 2 2.015 H

TABLE 56 MIC (μg/mL), FIC, FICI, for BisEDT in combination withAztreonam Organism: P. aeruginosa 9108 FICI (N): 5 Drug A: BisEDT Drug AMIC:  2 SUM FICI: 3.97 Drug B: Aztreonam Drug B MIC: 16 MEAN FICI: 0.79Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5  8 0.5 1 D 0.5 0.25 8 0.5 0.75 E 0.25 0.125  8 0.5 0.625 F 0.12 0.06  8 0.5 0.56 G 0.060.03 16 1 1.03 H

TABLE 57 MIC (μg/mL), FIC, FICI, for BisEDT in combination with ColistinOrganism: P. aeruginosa 9108 FICI (N): 6 Drug A: BisEDT Drug A MIC: 4SUM FICI: 6.48 Drug B: Colistin Drug B MIC: 0.5 MEAN FICI: 1.08 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B 2 0.5 0.25 0.5 1 C 1 0.25 0.5 11.25 D 0.5 0.125 0.5 1 1.125 E 0.25 0.0625 0.5 1 1.0625 F 0.12 0.03 0.51 1.03 G 0.06 0.015 0.5 1 1.015 H

TABLE 58 MIC (μg/mL), FIC, FICI, for BisEDT in combination withMeropenem Organism: B. cepacia 546 FICI (N): 5 Drug A: BisEDT Drug AMIC: 2 SUM FICI: 9.97 Drug B: Meropenem Drug B MIC: 4 MEAN FICI: 1.99Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 4 1 1.5 D 0.5 0.258 2 2.25 E 0.25 0.125 8 2 2.125 F 0.12 0.06 8 2 2.06 G 0.06 0.03 8 22.03 H

TABLE 59 MIC (μg/mL), FIC, FICI, for BisEDT in combination withTobramycin Organism: B. cepacia 546 FICI (N): 5 Drug A: BisEDT Drug AMIC:  2 SUM FICI: 3.47 Drug B: Tobramycin Drug B MIC: 32 MEAN FICI: 0.69Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 16 0.5 1 D 0.5 0.2516 0.5 0.75 E 0.25 0.125 16 0.5 0.625 F 0.12 0.06 16 0.5 0.56 G 0.060.03 16 0.5 0.53 H

TABLE 60 MIC (μg/mL), FIC, FICI, for BisEDT in combination with AmikacinOrganism: B. cepacia 546 FICI (N): 5 Drug A: BisEDT Drug A MIC: 2 SUMFICI: 3.22 Drug B: Amikacin Drug B MIC: 64 MEAN FICI: 0.64 Row MIC_(A)FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 16 0.25 0.75 D 0.5 0.25 32 0.50.75 E 0.25 0.125 32 0.5 0.625 F 0.12 0.06 32 0.5 0.56 G 0.06 0.03 320.5 0.53 H

TABLE 61 MIC (μg/mL), FIC, FICI, for BisEDT in combination withCiprofloxacin Organism: B. cepacia 546 FICI (N): 6 Drug A: BisEDT Drug AMIC: 4 SUM FICI: 8.11 Drug B: Ciprofloxacin Drug B MIC: 2 MEAN FICI:1.35 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B 2 0.5 0.25 0.125 0.625C 1 0.25 4 2 2.25 D 0.5 0.125 4 2 2.125 E 0.25 0.063 2 1 1.063 F 0.120.03 2 1 1.03 G 0.06 0.015 2 1 1.015 H

TABLE 62 MIC (μg/mL), FIC, FICI, for BisEDT in combination withAztreonam Organism: B. cepacia 546 FICI (N): 6 Drug A: BisEDT Drug AMIC:  4 SUM FICI: 6.98 Drug B: Aztreonam Drug B MIC: 32 MEAN FICI: 1.16Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B 2 0.5 32 1 1.5 C 1 0.25 321 1.25 D 0.5 0.125 32 1 1.125 E 0.25 0.063 32 1 1.063 F 0.12 0.03 32 11.03 G 0.06 0.015 32 1 1.015 H

TABLE 63 MIC (μg/mL), FIC, FICI, for BisEDT in combination with ColistinOrganism: B. cepacia 546 FICI (N): 6 Drug A: BisEDT Drug A MIC:    4 SUMFICI: 1.44 Drug B: Colistin Drug B MIC: >256 MEAN FICI: 0.24 Row MIC_(A)FIC_(A) MIC_(B) FIC_(B) FICI A B 2 0.5  0.25 0.0005 0.500 C 1 0.25  80.016 0.266 D 0.5 0.125 32 0.063 0.188 E 0.25 0.063 64 0.125 0.188 F0.12 0.03 64 0.125 0.155 G 0.06 0.015 64 0.125 0.14 H

TABLE 64 MIC (μg/mL), FIC, FICI, for BisEDT in combination withMeropenem Organism: B. cepacia 1793 FICI (N):  5 Drug A: BisEDT Drug AMIC: 2 SUM FICI: 12.97 Drug B: Meropenem Drug B MIC: 4 MEAN FICI:  2.59Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5  8 2 2.5 D 0.5 0.25 8 2 2.25 E 0.25 0.125 16 4 4.125 F 0.12 0.06  8 2 2.06 G 0.06 0.03  8 22.03 H

TABLE 65 MIC (μg/mL), FIC, FICI, for BisEDT in combination withTobramycin Organism: B. cepacia 1793 FICI (N): 4 Drug A: BisEDT Drug AMIC: 1 SUM FICI: 4.43 Drug B: Tobramycin Drug B MIC: 16 MEAN FICI: 1.11Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 8 0.5 1 E 0.250.25 16 1 1.25 F 0.12 0.12 16 1 1.12 G 0.06 0.06 16 1 1.06 H

TABLE 66 MIC (μg/mL), FIC, FICI, for BisEDT in combination with AmikacinOrganism: B. cepacia 1793 FICI (N): 5 Drug A: BisEDT Drug A MIC:  1 SUMFICI: 6.43 Drug B: Amikacin Drug B MIC: 32 MEAN FICI: 1.29 Row MIC_(A)FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 1 16 0.5 1.5 D 0.5 0.5 32 1 1.5 E0.25 0.25 32 1 1.25 F 0.12 0.12 32 1 1.12 G 0.06 0.06 32 1 1.06 H

TABLE 67 MIC (μg/mL), FIC, FICI, for BisEDT in combination withCiprofloxacin Organism: B. cepacia 1793 FICI (N): 4 Drug A: BisEDT DrugA MIC: 1 SUM FICI: 7.93 Drug B: Ciprofloxacin Drug B MIC: 0.5 MEAN FICI:1.98 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 1 2 2.5 E0.25 0.25 1 2 2.25 F 0.12 0.12 1 2 2.12 G 0.06 0.06 0.5 1 1.06 H

TABLE 68 MIC (μg/mL), FIC, FICI, for BisEDT in combination withAztreonam Organism: B. cepacia 1793 FICI (N): 4 Drug A: BisEDT Drug AMIC:  1 SUM FICI: 4.93 Drug B: Aztreonam Drug B MIC: 128 MEAN FICI: 1.23Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 128 1 1.5 E0.25 0.25 128 1 1.25 F 0.12 0.12 128 1 1.12 G 0.06 0.06 128 1 1.06 H

TABLE 69 MIC (μg/mL), FIC, FICI, for BisEDT in combination with ColistinOrganism: B. cepacia 1793 FICI (N): 4 Drug A: BisEDT Drug A MIC: 1 SUMFICI: 1.32 Drug B: Colistin Drug B MIC: >256 MEAN FICI: 0.33 Row MIC_(A)FIC_(A) MIC_(B) FIC_(B) FICI A B C D 0.5 0.5 8 0.016 0.516 E 0.25 0.2564 0.125 0.375 F 0.12 0.12 64 0.125 0.245 G 0.06 0.06 128 0.125 0.185 H

TABLE 70 MIC (μg/mL), FIC, FICI, for BisEDT in combination withMeropenem Organism: B. cenocepacia 548 FICI (N): 4 Drug A: BisEDT Drug AMIC: 4 SUM FICI: 4.44 Drug B: Meropenem Drug B MIC: 4 MEAN FICI: 1.11Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 2 0.5 2 0.5 1 E 1 0.254 1 1.25 F 0.5 0.125 4 1 1.125 G 0.25 0.063 4 1 1.063 H

TABLE 71 MIC (μg/mL), FIC, FICI, for BisEDT in combination withTobramycin Organism: B. cenocepacia 548 FICI (N): 4 Drug A: BisEDT DrugA MIC: 4 SUM FICI: 2.69 Drug B: Tobramycin Drug B MIC: 32 MEAN FICI:0.67 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 2 0.5 8 0.25 0.75E 1 0.25 16 0.5 0.75 F 0.5 0.125 16 0.5 0.625 G 0.25 0.063 16 0.5 0.563H

TABLE 72 MIC (μg/mL), FIC, FICI, for BisEDT in combination with AmikacinOrganism: B. cenocepacia 548 FICI (N): 4 Drug A: BisEDT Drug A MIC: 4SUM FICI: 2.69 Drug B: Amikacin Drug B MIC: 128 MEAN FICI: 0.67 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 2 0.5 32 0.25 0.75 E 1 0.2564 0.5 0.75 F 0.5 0.125 64 0.5 0.625 G 0.25 0.063 64 0.5 0.563 H

TABLE 73 MIC (μg/mL), FIC, FICI, for BisEDT in combination withCiprofloxacin Organism: B. cenocepacia 548 FICI (N): 4 Drug A: BisEDTDrug A MIC: 4 SUM FICI: 12.94 Drug B: Ciprofloxacin Drug B MIC: 2 MEANFICI: 3.23 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 2 0.5 8 44.5 E 1 0.25 8 4 4.25 F 0.5 0.125 4 2 2.125 G 0.25 0.063 4 2 2.063 H

TABLE 74 MIC (μg/mL), FIC, FICI, for BisEDT in combination withAztreonam Organism: B. cenocepacia 548 FICI (N): 4 Drug A: BisEDT Drug AMIC: 4 SUM FICI: 4.94 Drug B: Aztreonam Drug B MIC: 64 MEAN FICI: 1.23Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 2 0.5 64 1 1.5 E 1 0.2564 1 1.25 F 0.5 0.125 64 1 1.125 G 0.25 0.063 64 1 1.063 H

TABLE 75 MIC (μg/mL), FIC, FICI, for BisEDTin combination with ColistinOrganism: B. cenocepacia 548 FICI (N): 4 Drug A: BisEDT Drug A MIC: 4SUM FICI: 1.63 Drug B: Colistin Drug B MIC: >256 MEAN FICI: 0.41 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C D 2 0.5 32 0.063 0.563 E 10.25 64 0.125 0.375 F 0.5 0.125 128 0.25 0.375 G 0.25 0.063 128 0.250.313 H

TABLE 76 MIC (μg/mL), FIC, FICI, for BisEDT in combination withMeropenem Organism: B. cenocepacia 813 FICI (N): 6 Drug A: BisEDT Drug AMIC: 4 SUM FICI: 6.48 Drug B: Meropenem Drug B MIC: 2 MEAN FICI: 1.08Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B 2 0.5 1 0.5 1 C 1 0.25 2 11.25 D 0.5 0.125 2 1 1.125 E 0.25 0.063 2 1 1.063 F 0.12 0.03 2 1 1.03 G0.06 0.015 2 1 1.015 H

TABLE 77 MIC (μg/mL), FIC, FICI, for BisEDT in combination withTobramycin Organism: B. cenocepacia 813 FICI (N): 6 Drug A: BisEDT DrugA MIC: 4 SUM FICI: 5.48 Drug B: Tobramycin Drug B MIC: 256 MEAN FICI:0.91 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B 2 0.5 128 0.5 1 C 10.25 128 0.5 0.75 D 0.5 0.125 128 0.5 0.625 E 0.25 0.063 256 1 1.063 F0.12 0.03 256 1 1.03 G 0.06 0.015 256 1 1.015 H

TABLE 78 MIC (μg/mL), FIC, FICI, for BisEDT in combination with AmikacinOrganism: B. cenocepacia 813 FICI (N): 5 Drug A: BisEDT Drug A MIC: 2SUM FICI: 4.97 Drug B: Amikacin Drug B MIC: >256 MEAN FICI: 0.99 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 256 0.5 1 D 0.5 0.25256 0.5 0.75 E 0.25 0.125 >256 1 1.125 F 0.12 0.06 >256 1 1.06 G 0.060.03 >256 1 1.03 H

TABLE 79 MIC (μg/mL), FIC, FICI, for BisEDT in combination withCiprofloxacin Organism: B. cenocepacia 813 FICI (N): 6 Drug A: BisEDTDrug A MIC: 4 SUM FICI: 17.11 Drug B: Ciprofloxacin Drug B MIC: 2 MEANFICI: 2.85 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B 2 0.5 0.25 0.1250.625 C 1 0.25 8 4 4.25 D 0.5 0.125 8 4 4.125 E 0.25 0.063 8 4 4.063 F0.12 0.03 4 2 2.03 G 0.06 0.015 4 2 2.015 H

TABLE 80 MIC (μg/mL), FIC, FICI, for BisEDT in combination withAztreonam Organism: B. cenocepacia 813 FICI (N): 5 Drug A: BisEDT Drug AMIC: 2 SUM FICI: 5.97 Drug B: Aztreonam Drug B MIC: 32 MEAN FICI: 1.19Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 32 1 1.5 D 0.5 0.2532 1 1.25 E 0.25 0.125 32 1 1.125 F 0.12 0.06 32 1 1.06 G 0.06 0.03 32 11.03 H

TABLE 81 MIC (μg/mL), FIC, FICI, for BisEDT in combination with ColistinOrganism: B. cenocepacia 813 FICI (N): 5 Drug A: BisEDT Drug A MIC: 2SUM FICI: 3.59 Drug B: Colistin Drug B MIC: >256 MEAN FICI: 0.72 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 64 0.125 0.625 D 0.50.25 256 0.5 0.75 E 0.25 0.125 256 0.5 0.625 F 0.12 0.06 256 0.5 0.56 G0.06 0.03 >256 1 1.03 H

TABLE 82 MIC (μg/mL), FIC, FICI, for BisEDT in combination withMeropenem Organism: B. multivorans 1795 FICI (N): 6 Drug A: BisEDT DrugA MIC: 2 SUM FICI: 7.97 Drug B: Meropenem Drug B MIC: 4 MEAN FICI: 1.33Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B 2 1 4 1 2 C 1 0.5 4 1 1.5 D0.5 0.25 4 1 1.25 E 0.25 0.125 4 1 1.125 F 0.12 0.06 4 1 1.06 G 0.060.03 4 1 1.03 H

TABLE 83 MIC (μg/mL), FIC, FICI, for BisEDT in combination withTobramycin Organism: B. multivorans 1795 FICI (N): 6 Drug A: BisEDT DrugA MIC: 2 SUM FICI: 7.97 Drug B: Tobramycin Drug B MIC: >256 MEAN FICI:1.33 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B 2 1 >256 1 2 C 10.5 >256 1 1.5 D 0.5 0.25 >256 1 1.25 E 0.25 0.125 >256 1 1.125 F 0.120.06 >256 1 1.06 G 0.06 0.03 >256 1 1.03 H

TABLE 84 MIC (μg/mL), FIC, FICI, for BisEDT in combination with AmikacinOrganism: B. multivorans 1795 FICI (N): 6 Drug A: BisEDT Drug A MIC: 2SUM FICI: 7.97 Drug B: Amikacin Drug B MIC: >256 MEAN FICI: 1.33 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B 2 1 >256 1 2 C 1 0.5 >256 1 1.5D 0.5 0.25 >256 1 1.25 E 0.25 0.125 >256 1 1.125 F 0.12 0.06 >256 1 1.06G 0.06 0.03 >256 1 1.03 H

TABLE 85 MIC (μg/mL), FIC, FICI, for BisEDT in combination withCiprofloxacin Organism: B. multivorans 1795 FICI (N): 5 Drug A: BisEDTDrug A MIC: 2 SUM FICI: 5.97 Drug B: Ciprofloxacin Drug B MIC: 32 MEANFICI: 1.19 Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 32 1 1.5D 0.5 0.25 32 1 1.25 E 0.25 0.125 32 1 1.125 F 0.12 0.06 32 1 1.06 G0.06 0.03 32 1 1.03 H

TABLE 86 MIC (μg/mL), FIC, FICI, for BisEDT in combination withAztreonam Organism: B. multivorans 1795 FICI (N): 5 Drug A: BisEDT DrugA MIC: 2 SUM FICI: 6.97 Drug B: Aztreonam Drug B MIC: 16 MEAN FICI: 1.39Row MIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B C 1 0.5 16 1 1.5 D 0.5 0.2516 1 1.25 E 0.25 0.125 16 1 1.125 F 0.12 0.06 16 1 1.06 G 0.06 0.03 32 22.03 H

TABLE 87 MIC (μg/mL), FIC, FICI, for BisEDT in combination with ColistinOrganism: B. multivorans 1795 FICI (N): 6 Drug A: BisEDT Drug A MIC: 2SUM FICI: 7.97 Drug B: Colistin Drug B MIC: >256 MEAN FICI: 1.33 RowMIC_(A) FIC_(A) MIC_(B) FIC_(B) FICI A B 2 >256 1 2 C 1 0.5 >256 1 1.5 D0.5 0.25 >256 1 1.25 E 0.25 0.125 >256 1 1.125 F 0.12 0.06 >256 1 1.06 G0.06 0.03 >256 1 1.03 H

REFERENCES

1.) Clinical and Laboratory Standards Institute (CLSI). PerformanceStandards for Antimicrobial Susceptibility Testing. 28th ed. CLSIsupplement M100. CLSI, 950 West Valley Road, Suite 2500, Wayne, Pa.19087 USA, 2018.

2.) CLSI. Methods for Dilution Antimicrobial Susceptibility Tests forBacteria That Grow Aerobically; Approved Standard—11^(th) Edition. CLSIstandard M07. CLSI, 950 West Valley Road, Suite 2500, Wayne, Pa. 19087USA, 2018.

3.) Eliopoulos G and R Moellering. 1991. Antimicrobial combinations. InAntibiotics in Laboratory Medicine, Third Edition, edited by V Lorian.Williams and Wilkins, Baltimore, Md., pp. 432-492.

4.) Odds FC. 2003. Synergy, antagonism, and what the chequerboard putsbetween them. J Antimicrob Chemother 52(1):1.

Example 11 In Vitro Activity of Bismuth Thiols and Comparators AgainstDrug Resistant Gram-Positive and -Negative Bacteria and Yeast

Introduction: The in vitro activity of three Bismuth Thiol compoundswere evaluated against organisms currently identified by the Centers forDisease Control (CDC; 1) as top drug-resistant threats in the UnitedStates, including ESKAPE pathogens (2, 3), C. difficile, resistantgonococci, and azole-resistant Candida spp. The susceptibility of testisolates to the Bismuth Thiol compounds MB-1-B3, MB-2B, and MB-6 andrelevant comparators was evaluated in accordance with guidelines fromthe Clinical and Laboratory Standards Institute (CLSI; 4-8).

Materials and Methods

Test and Comparator Agents: The test agents were stored at roomtemperature until assayed. All test agents were suspended and diluted in100% dimethylsulfoxide (DMSO), and were ultimately tested at a finalconcentration of 0.06-64 μg/mL. The stock solutions were allowed tostand for at least 1 hr prior to use to auto-sterilize.

Comparator drugs were tested over a concentration range spanningestablished quality control ranges and breakpoints. Information oncomparator compounds used during testing are described in Table 88below:

TABLE 88 Comparator Comounds Drug Manufacturer Lot No. Solvent DiluentLevofloxacin Sigma BCBC2112V 0.1M Water NaOH Meropenem USP I0J244 WaterWater Ceftazidime USP L1K237 Water Water Gentamicin Sigma SLBM9736VWater Water Vancomycin Water Water Penicillin Sigma 071M0740V WaterWater Oxacillin Sigma BCBF5635V Water Water Clindamycin Sigma 021M1533VWater Water Erythromycin Sigma 011M1510V 95% Water ethanol CiprofloxacinUSP 1134335 Water Water Metronidazole Sigma 095K0693 DMSO WaterFidaxomicin Merck SE-B13-01- DMSO Water 001885 Fluconazole USP H1L308DMSO DMSO Amphotericin B Sigma 063M4043V DMSO DMSO Trimethoprim Sigma080M4044 Water Water Sulfamethoxazole Fluka BCBC7096V Water and Water2.5M NaOH dropwise Ceftriaxone USP J1L040 Water Water

Test Organisms: The test organisms consisted of reference strains fromthe American Type Culture Collection (ATCC; Manassas, Va.) or clinicalisolates from the MMX repository. The spectrum of organisms evaluatedand their corresponding phenotypic information is shown in Tables 89-95.Relevant quality control organisms were included on each day of testingas specified by CLSI (4-8). The isolates were sub-cultured onto anappropriate agar medium prior to testing.

Test Media: Test media were prepared and stored in accordance withguidelines from CLSI (4, 6, 7). Broth microdilution susceptibilitytesting of aerobic bacteria was performed using cation adjustedMueller-Hinton Broth (CAMHB; Becton Dickinson [BD], Sparks, Md.; Lot No.6117994) with the exception of streptococci where CAMHB was supplementedwith 5% (v/v) lysed horse blood (Cleveland Scientific, Bath, OH; Lot No.322799). Neisseria gonorrhoeae were evaluated by agar dilution usingagar consisting of GC medium base (BD; Lot No. 4274618) supplementedwith 1% IsoVitaleX (BD; Lot No. 5246843).

The susceptibility of anaerobic bacteria was determined by agar dilutionusing Brucella Agar (BD/BBL; Lot No. 5237692) supplemented with 5 μg/mLhemin (Sigma, St. Louis, Mo.; Lot No. 108K1088), 1 μg/mL Vitamin K1 and5% (v/v) laked sheep blood (Cleveland Scientific, Bath, Ohio; Lot No.322799).

The susceptibility of yeast isolates was determined by brothmicrodilution in RPMI medium (HyClone Laboratories, Logan, UT; Lot No.AZC184041B) buffered with 0.165 M MOPS (Calbiochem, Billerica, Mass.;Lot No. 2694962). The pH of the medium was adjusted to 7.0 with 1 NNaOH, sterile filtered using a 0.2 μm PES filter, and stored at 4° C.until used.

Broth Microdilution MIC Testing (Aerobic Bacteria and Yeast): The brothmicrodilution assay method employed for the susceptibility testing ofaerobic bacteria (excluding N. gonorrhoeae which was evaluated by agardilution) and yeast essentially followed the procedures described byCLSI (3, 4, 7, 8) and employed automated liquid handlers to conductserial dilutions and liquid transfers. Automated liquid handlersincluded the Multidrop 384 (Lab systems, Helsinki, Finland) and Biomek2000 (Beckman Coulter, Fullerton Calif.). The wells in columns 2-12 instandard 96-well microdilution plates (Costar 3795) were filled with 150μl of the correct diluent. These would become the ‘mother plates’ fromwhich ‘daughter’ or test plates would be prepared. The drugs (300 μL at40× the desired top concentration in the test plates) were dispensedinto the appropriate well in Column 1 of the mother plates. The Biomek2000 was used to make serial two-fold dilutions through Column 11 in the“mother plate”. The wells of Column 12 contained no drug and ultimatelyserved as the organism growth control wells.

The daughter plates were loaded with 185 μL per well of the appropriatetest media using the Multidrop 384. The daughter plates were preparedusing the Biomek FX which transferred 5 μL of drug solution from eachwell of a mother plate to the corresponding well of the correct daughterplate in a single step.

A standardized inoculum of each organism was prepared per CLSI methods(3, 7). Isolated colonies of each test isolate were picked from theprimary plate and a suspension was prepared to equal a 0.5 McFarlandturbidity standard. Standardized suspensions were then diluted 1:100 intest media (1:100 for yeast, 1:20 for bacteria). After dilution, theinoculum suspensions were then transferred to compartments of sterilereservoirs divided by length (Beckman Coulter), and the Biomek 2000 wasused to inoculate all plates. Daughter plates were placed on the Biomek2000 in reverse orientation so that plates were inoculated from low tohigh drug concentration.

The Biomek 2000 delivered 10 μL of standardized inoculum into each wellof the appropriate daughter plate for an additional 1:20 dilution. Thewells of the daughter plates ultimately contained 185 μL of theappropriate media, 5 μL of drug solution, and 10 μL of inoculum whichcorresponded to a final inoculum concentration of 0.5-2.5×103 CFU/mL ofyeast and approximately 5×10⁵ CFU/mL of bacteria per test well. Thefinal concentration of DMSO (if used as a solvent) in the test well was2.5%.

Plates were stacked 4 high, covered with a lid on the top plate, placedinto plastic bags, and incubated at 35° C. for approximately 24-48 hrfor all yeast isolates and 16-24 hr for aerobic bacteria. Plates wereviewed from the bottom using a plate viewer. An un-inoculated solubilitycontrol plate was observed for evidence of drug precipitation. MICendpoints for the test agents and control compounds were read per CLSIcriteria (3, 7).

Agar Dilution MIC Testing (Anaerobic Bacteria and Gonococci): MIC valuesfor anaerobic bacteria were determined using a reference agar dilutionmethod as described by CLSI (6). Organisms were grown at 35° C. in theBactron II Anaerobic Chamber (Shel Lab, Cornelius, OR) for approximately48 hr prior to the assay. Drug dilutions and drug-supplemented agarplates were prepared manually per CLSI (6). The plates were allowed tostand at room temperature for 1 hr to allow the agar surface to dry andpre-reduced for approximately 1 hr in the anaerobe chamber prior toinoculation. Each isolate was suspended to the equivalent of a 0.5McFarland standard in Brucella broth using a turbidity meter (DadeBehring MicroScan, West Sacramento, Calif.). Each bacterial cellsuspension was then diluted 1:10 in Brucella broth and transferred towells in a stainless steel replicator block which was used to inoculatethe test plates. The prongs on the replicator deliver approximately 1-2μl of inoculum to an agar surface. The resulting inoculum spotscontained approximately 1×10⁵ CFU/spot. After the inoculum dried, theinoculated drug-supplemented agar plates and no drug growth controlplates were incubated at 35° C. for 42-48 hr in the anaerobe chamber.The MIC was read per CLSI guidelines (6).

MIC values for N. gonorrhoeae were determined using the reference agardilution method as described by CLSI (4). This method followed the sameagar dilution method described above for anaerobes with the exceptionthat agar plates contained GC medium base supplemented with 1%IsoVitaleX and, after inoculation, plates were incubated aerobically at35° C. in 5% CO2 for 20-24 hr.

Results and Discussion

The activity of the Bismuth Thiol test agents MB-1-B3, MB-2B, and MB-6and comparators are shown below for Enterobacteriaceae (Table 89),Pseudomonas aeruginosa and Acinetobacter baumannii (Table 90),Staphylococcus aureus and Enterococcus spp. (Table 91), streptococci(Table 92), N. gonorrhoeae (Table 93), anaerobes (Table 94), and Candidaspp. (Table 95). Across all evaluated organisms, MIC results forcomparator agents were within the established CLSI QC ranges for therelevant ATCC QC isolate (5, 8).

The evaluated Enterobacteriaceae (Table 1) consisted of the Escherichiacoli ATCC QC isolate, ESBL-positive E. coli and Klebsiella pneumoniae,KPC-positive K. pneumoniae, and NDM-1 positive E. coli, K. pneumoniae,and Enterobacter cloacae. Excluding the ATCC QC isolate of E. coli whichwas susceptible, the activity of the comparators illustrates thedrug-resistant nature of these isolates. Regardless of the high degreeof drug resistance, the evaluated Bismuth Thiol test agents hadconsistent activity across all isolates. MB-1-B3 (MIC values of 0.5-4μg/mL) and MB-6 (MIC values of 0.5-2 μg/mL) had similar activity andthis activity was typically 2- to 16-fold lower than that observed withMB-2B (MIC values of 2-32 μg/mL).

The evaluated P. aeruginosa and A. baumannii (Table 90) consisted of thesusceptible P. aeruginosa ATCC QC isolate, and various isolates witheither metallo-beta-lactamases or multi-drug resistance. Excluding theQC isolate, the activity of comparators illustrates the drug-resistantnature of these isolates. Regardless of the high degree of drugresistance, the evaluated Bismuth Thiol test agents had consistentactivity across all isolates. MB-1-B3 and MB-6 (MIC values of 0.5-2μg/mL) had similar activity and this activity was typically 2- to32-fold lower than that observed with MB-2B (MIC values of 2-16 μg/mL).

Against S. aureus and Enterococcus spp. (Table 91), all 3 Bismuth Thioltest compounds had potent activity regardless of resistance phenotype(MRSA for S. aureus and VRE for E. faecalis and E. faecium). Theevaluated MRSA were largely susceptible to vancomycin and gentamicin butresistant to the remaining comparators. Regardless of resistance,MB-1-B3 and MB-6 had MIC values of <0.06 μg/mL against MRSA and MB-2Balso had MIC values of <0.06 μg/mL with the exception of the QC isolateand MRSA MMX 9203 (MIC values of 0.5 and 0.25 μg/mL, respectively).Against enterococci, there was little activity observed with theevaluated comparators. The Bismuth Thiol test agents were active thoughwith slightly higher MIC values for vancomycin-resistant E. faeciumrelative to vancomycin-resistant E. faecalis. As with the Gram-negativeaerobic isolates, for enterococci MB-1-B3 (MIC values of 0.25-2 μg/mL)and MB-6 (MIC values of 0.25-1 μg/mL) had similar activity and thisactivity was typically 4- to 8-fold lower than that observed with MB-2B(MIC values of 2-4 μg/mL).

The evaluated streptococci (Table 92) consisted of the susceptible S.pneumoniae QC isolate, multi-drug resistant pneumococci,macrolide-resistant S. pyogenes, and clindamycin-resistant S.agalactiae. Regardless of drug-resistance phenotype, the bismuth-thioltest agents maintained activity against streptococci. Among the 3Bismuth Thiol test agents, there was trend towards slightly higher MICvalues against pneumococci relative to beta-hemolytic streptococci forMB-1-B3 and MB-6. Against pneumococci, MB-1-B3 (MIC values of 0.5-1μg/mL) and MB-6 (MIC values of 0.5-8 μg/mL) had similar activity andthis activity was typically 8- to 16-fold lower than that observed withMB-2B (MIC values of 1-8 μg/mL). Against beta-hemolytic streptococci,MB-1-B3 (MIC values of 0.03-1μg/mL) and MB-6 (MIC values of 0.03-2μg/mL)had similar activity and this activity was typically 4- to 16-fold lowerthan that observed with MB-2B (MIC values of 0.25-8 μg/mL).

As shown in Table 93, the Bismuth Thiol test agents had potent activityagainst the susceptible QC isolate of N. gonorrhoeae, the 3ciprofloxacin-resistant isolates, and the single ceftriaxonenon-susceptible isolate. Similar activity was observed with MB-1-B3 (MICvalues of 0.06-0.12 μg/mL) and MB-6 (MIC values of 0.06-0.25 μg/mL) andthis activity was slightly greater than that observed for MB-2B (MICvalues of 0.12-0.5 μg/mL).

Against the evaluated anaerobes (Table 94) which consisted of thesusceptible Bacteroides fragilis and Clostridium difficile QC isolatesand C. difficile with various clinically important ribotypes including027 (hypervirulent strain), MB-1-B3 (MIC values of 0.25-2μg/mL) and MB-6(MIC values of 1-4 μg/mL) had similar activity and this activity wastypically slightly greater than that observed with MB-2B (MIC values of2-16 μg/mL). Resistance to comparators clindamycin, metronidazole, andfidaxomicin appeared to have no impact of the activity of the BismuthThiol test agents.

Finally, against azole-resistant isolates of clinically prevalentCandida spp. (Table 95) including C. parapsilosis, C. albicans, C.glabrata, and C. tropicalis, the Bismuth Thiol test agents were active.A trend towards higher MIC values for the test agents was observed withC. albicans and C. tropicalis relative to C. parapsilosis and C.glabrata. All 3 Bismuth Thiol test agents had similar activity againstyeast, with MIC values of 0.25-0.5 μg/mL at 24 hr against C.parapsilosis and C. glabrata, 1-4 μg/mL against C. albicans, and 1-16μg/mL against C. tropicalis.

In summary, the broad spectrum activity of the Bismuth Thiol test agentsevaluated in this study was clear and the activity observed againstsusceptible QC isolates was maintained against drug resistant isolatesregardless of the organism or resistance phenotype evaluated. TheBismuth Thiol test agents were the most active against MRSA, N.gonorrhoeae, and beta-hemolytic streptococci based on MIC values butwere also highly active against Gram-negative aerobes, S. pneumoniae, C.difficile, and yeast. In general, test agents MB-1-B3 and MB-6 hadsimilar activity by MIC and both were more potent than MB-2B, with theexception of yeast and to a lesser extent N. gonorrhoeae where all 3compounds had similar activity profiles.

TABLE 89 Minimal Inhibitory Concentration (MIC) Values for MicrobionBismuth Thiol Test Agents and Comparators Against Enterobacteriaceae MIC(μg/mL) Isolate Type MB-1-B3 MB-2B MB-6 Levofloxacin CiprofloxacinMeropenem Ceftazidime Gentamicin E. coli QC 0.5 2 0.5 0.015 0.008 0.030.12 1 MMX 102 (0.008-0.06)¹ (0.004-0.015) (0.008-0.06) (0.06-0.5)(0.25-1) (ATCC 25922) E. coli ESBL 1 2 1 16 32 0.03 32 64 MMX 8423Lev^(R) CAZ^(R) Gm^(R) E. coli ESBL 0.5 4 0.5 0.06 0.015 0.03 16 0.25MMX 8424 CAZ^(R) E. coli ESBL 1 2 1 16 >64 0.015 16 >64 MMX 8425 Lev^(R)CAZ^(R) Gm^(R) E. coli NDM-1 1 2 1 16 >64 32 >32 >64 MMX 5980 Lev^(R)(ATCC BAA-2469) MEM^(R) CAZ^(R) Gm^(R) K. pneumoniae KPC-2 1 8 1 >64 >6432 >32 1 MMX 4683 Lev^(R) MEM^(R) CAZ^(R) K. pneumoniae KPC-2 2 16 1 10.03 >64 >32 0.25 MMX 4622 MEM^(R) CAZ^(R) K. pneumoniae KPC-2 2 32 0.51 2 >64 >32 64 MMX 4623 MEM^(R) CAZ^(R) GM^(R) K. pneumoniae KPC-3 2 8 164 >64 32 >32 8 MMX 4694 Lev^(R) MEM^(R) CAZ^(R) K. pneumoniae KPC-3 416 2 64 >64 >64 >32 1 MMX 4653 Lev^(R) MEM^(R) CAZ^(R) K. pneumoniaeESBL 4 32 2 0.03 0.5 8 8 0.25 MMX 4684 MEM^(R) K. pneumoniae ESBL 2 8 132 64 4 >32 1 MMX 4685 Lev^(R) MEM^(R) CAZ^(R) K. pneumoniae NDM-1 2 161 >64 >64 >64 >32 >64 MMX 5979 Lev^(R) MEM^(R) CAZ^(R) Gm^(R) E. cloacaeNDM-1 4 32 2 64 >64 >64 >32 >64 MMX 5981 Lev^(R) (ATCC BAA-2468) MEM^(R)CAZ^(R) Gm^(R) QC = quality control; ESBL = extended-spectrumbeta-lactamase; KPC = K. pneumoniae carbapenemase; NDM = New DelhiMetallo-beta-lactamase; Lev^(R) = Levofloxacin-resistant; MEM^(R) =Meropenem-resistant; CAZ^(R) = ceftazidime-resistant; Gm^(R) =Gentamicin-resistant ¹ CLSI QC ranges shown in parenthesis whereapplicable

TABLE 90 Minimal Inhibitory Concentration (MIC) Values for MicrobionBismuth Thiol Test Agents and Comparators Against P. aeruginosa and A.baumannii MI Isolate Type MB-1-B3 MB-2B MB-6 Levofloxacin CiprofloxacinMeropenem Ceftazidime Gentamicin P. aeruginosa QC 1 8 1 1 0.5 0.5 2 1MMX 103 (0.5-4) (0.25-1) (0.25-1) (1-4) (0.5-2) (ATCC 27853) P.aeruginosa VIM-2 1 16 2 32 32 8 32 4 MMX 4697 Lev^(R) Cip^(R) MEM^(R)CAZ^(R) P. aeruginosa IMP-7 2 8 2 64 32 >64 >32 >64 MMX 4654 Lev^(R)Cip^(R) MEM^(R) CAZ^(R) P. aeruginosa MDR 1 4 2 64 64 32 >32 8 MMX 2562Lev^(R) Cip^(R) MEM^(R) CAZ^(R) P. aeruginosa MDR 1 2 1 64 32 16 32 8MMX 1381 Lev^(R) Cip^(R) MEM^(R) CAZ^(R) P. aeruginosa MDR 1 8 2 64 3216 16 4 MMX 3991 Lev^(R) Cip^(R) MEM^(R) CAZ^(R) A. baumannii MDR; 0.516 0.5 8 32 64 >32 8 MMX 4651 OXA-27 (NCTC 13304) Lev^(R) Cip^(R)MEM^(R) CAZ^(R) Gm^(I) A. baumannii MDR 0.5 16 0.5 64 >64 64 32 >64 MMX2592 Lev^(R) Cip^(R) MEM^(R) CAZ^(R) Gm^(R) A. baumannii MDR 1 16 0.532 >64 64 32 >64 MMX 2593 Lev^(R) Cip^(R) MEM^(R) CAZ^(R) Gm^(R) A.baumannii MDR 1 16 1 1 4 4 16 0.5 MMX 3372 Cip^(R) MEM^(I) CAZ^(I) A.baumannii Sensitive 1 16 0.5 0.12 0.5 0.5 4 0.12 MMX 3373 QC = qualitycontrol; VIM/IMP = metallo-beta lactamase type; OXA = type Dextended-spectrum beta-lactamase; MDR = multi-drug resistant (based onresistance to at least 3 different classes of antibiotic); Lev^(R) =levofloxacin-resistant; CIP^(R) = Ciprofloxacin-resistant; MEM^(I) =meropenem intermediate resistance; MEM^(R) = meropenem-resistant;CAZ^(R) = ceftazidime-resistant; CAZ^(I) = ceftazidime intermediateresistance; Gm^(R) = gentamicin-resistant; Gm^(I) = gentamicinintermediate resistance. ¹ CLSI QC ranges shown in parenthesis whereapplicable

TABLE 91 Minimal Inhibitory Concentration (MIC) Values for MicrobionBismuth Thiol Test Agents and Comparators Against S. aureus andEnterococcus spp. MIC (μg/mL) Levo- Cipro- Genta- Vanco- Mero- Ceftaz-Clind- Eryth- Oxa- Isolate Type MB-1-B3 MB-2B MB-6 floxacin floxacinmicin mycin penem idime amycin romycin cillin S. aureus QC; ≤0.06 0.5≤0.06 0.12 0.5 0.12 1 0.06 8 0.12 0.5 0.25 MMX 101 MSSA (0.06- (0.12-(0.12-1) (0.5-2) (0.03- (4-16) (0.06- (0.25-1) (0.12- (ATCC 29213) 0.5)¹0.5) 0.12) 0.25) 0.25) S. aureus MRSA² ≤0.06 0.25 ≤0.06 4 16 0.12 24 >32 0.12 >64 64 MMX 9203 Lev^(R) Cip^(R) Ery^(R) Ox^(R) S. aureus MRSA≤0.06 ≤0.06 ≤0.06 4 16 0.25 1 4 >32 0.12 >64 64 MMX 9204 Lev^(R) Cip^(R)Ery^(R) Ox^(R) S. aureus MRSA ≤0.06 ≤0.06 ≤0.06 >64 >64 0.25 264 >32 >32 >64 >64 MMX 9205 Lev^(R) Cip^(R) CC^(R) Ery^(R) Ox^(R) S.aureus MRSA ≤0.06 ≤0.06 ≤0.06 32 >64 0.25 2 32 >32 >32 >64 >64 MMX 5373Lev^(R) Cip^(R) CC^(R) Ery^(R) Ox^(R) S. aureus MRSA ≤0.06 ≤0.06 ≤0.0632 >64 0.25 0.25 8 >32 >32 >64 32 MMX 6311 Lev^(R) Cip^(R) CC^(R)Ery^(R) Ox^(R) E. faecalis VRE 0.5 2 0.5 32 64 >64 >64 8 >32 >32 >64 >64MMX 8960 Lev^(R) Cip^(R) Ery^(R) E. faecalis VRE 0.25 2 0.25 64 64 8 >648 >32 >32 >64 64 MMX 8961 Lev^(R) Cip^(R) Ery^(R) E. faecalis VRE 0.5 20.25 64 64 >64 >64 2 >32 >32 >64 16 MMX 8962 Lev^(R) Cip^(R) Ery^(R) E.faecium VRE 2 4 1 >64 >64 >64 >64 >64 >32 >32 >64 >64 MMX 752 vanALev^(R) Cip^(R) Ery^(R) E. faecium VRE 1 20.5 >64 >64 >64 >64 >64 >32 >32 >64 >64 MMX 485 vanA Lev^(R) Cip^(R)Ery^(R) QC = quality control; MSSA = methicillin-susceptible S. aureus;MRSA = methicillin-resistant S. aureus; VRE = vancomycin-resistantenterococci; vanA = vanA-type VRE (based on vancomycin- andteicoplanin-resistant phenotype); Lev^(R) = levofloxacin-resistant;CIP^(R) = Ciprofloxacin-resistant; CC^(R) = Clindamycin-resistant;Ery^(R) = Erythromycin-resistant; Ox^(R) = oxacillin-resistant ¹CLSI QCranges shown in parenthesis where applicable ²MRSA do not havebreakpoints for ceftazidime and meropenem - resistance is presumed.

TABLE 92 Minimal Inhibitory Concentration (MIC) Values for MicrobionBismuth Thiol Test Agents and Comparators Against Streptococcus spp. MIC(μg/mL) MB-1- MB- MB- Levo- Cipro- Mero- Vanco- Trimeth/ Clinda- Eryth-Peni- Isolate Type B3 2B 6 floxacin floxacin penem mycin Sulfa mycinromycin cillin S. pneumoniae QC 0.5 8 0.5 0.5 0.5 0.06 0.25 0.25/4.80.06 0.03 0.25 MMX 6837 (0.5-2)¹ (0.06-0.25) (0.12-0.5) (0.12/2.4-1/19)(0.03-0.12) (0.03-0.12) (0.25-1) (ATCC 49619) S. pneumoniae Lev^(R) 1 80.5 8 32 0.5 0.5  4/76 >8 >8 2 MMX 880 SXT^(R) CC^(R) Ery^(R) Pen^(R) S.pneumoniae Mem^(R) 1 1 8 1 1 1 0.5  16/304 >8 >8 4 MMX 937 SXT^(R)CC^(R) Ery^(R) Pen^(R) S. pneumoniae CC^(R) 1 8 1 1 4 0.03 0.5 2/38 >8 >8 0.12 MMX 3959 Ery^(R) S. pneumoniae Mem^(R) 1 8 0.5 1 2 10.25  16/304 >8 >8 4 MMX 5440 SXT^(R) CC^(R) Ery^(R) Pen^(R) S.pneumoniae Mem^(R) 0.5 8 0.5 1 2 1 0.25   8/152 >8 >8 4 MMX 5445 SXT^(R)CC^(R) Ery^(R) Pen^(R) S. pneumoniae Mem^(R) 0.25 8 0.5 1 1 1 0.25  8/152 >8 >8 4 MMX 8133 SXT^(R) CC^(R) Ery^(R) Pen^(R) S. pyogenesEry^(R) 0.12 2 0.12 0.5 0.5 ≤0.008 0.5 0.12/2.4 0.06 >8 ≤0.008 MMX 3068S. pyogenes ERY^(R) 0.25 1 0.5 0.25 0.12 ≤0.008 0.5 0.12/2.4 1 >8 ≤0.008MMX 3820 CLI^(R) S. pyogenes ERY^(R) 0.03 0.25 0.03 0.12 0.25 ≤0.008 0.50.06/1.2 0.12 >8 ≤0.008 MMX 3919 S. pyogenes ERY^(R) 0.25 1 0.5 0.5 0.5≤0.008 0.5 0.25/4.8 >8 >8 ≤0.008 MMX 3929 CC^(R) S. pyogenes ERY^(R) 0.51 1 0.25 0.25 ≤0.008 0.5 0.06/1.2 >8 >8 ≤0.008 MMX 5091 CC^(R) S.agalactiae ERY^(R) 0.5 4 0.25 0.5 0.5 0.03 0.5 0.12/2.4 >8 8 0.03 MMX3741 CC^(R) S. agalactiae ERY^(R) 0.25 4 0.25 1 1 0.06 0.50.12/2.4 >8 >8 0.06 MMX 3743 CC^(R) S. agalactiae ERY^(R) 0.5 4 0.25 0.51 0.06 0.5 0.12/2.4 >8 >8 0.06 MMX 4077 CC^(R) S. agalactiae ERY^(R) 1 80.5 1 1 8 2 0.12/2.4 >8 >8 2 MMX 4079 CC^(R) Pen^(R) MEM^(R) S.agalactiae ERY^(R) 0.25 2 0.25 0.5 0.5 0.06 1 0.12/2.4 >8 >8 0.06 MMX4086 CC^(R) QC = quality control; Trimeth = trimethoprim; Sulfa =sulfamethoxazole; MDR = multi-drug resistant (based on resistance to atleast 3 different classes of antibiotic); Ery^(R) =erythromycin-resistant; CC^(R) = clindamycin-resistant; SXT^(R) =Trimethoprim/Sulfamethoxazole-resistant; MEM^(R) = Meropenem-resistant;Pen^(R) = Penicillin-resistant ¹CLSI QC ranges shown in parenthesiswhere applicable

TABLE 93 Minimal Inhibitory Concentration (MIC) Values for MicrobionBismuth Thiol Test Agents and Comparators Against N. gonorrhoeae MIC(μg/mL) Isolate Type MB-1-B3 MB-2B MB-6 Ciprofloxacin Ceftriaxone N.gonorrhoeae QC 0.06 0.12 0.06 0.008 0.015 MMX 683 (0.001-0.008)¹(0.004-0.015) (ATCC 49226) N. gonorrhoeae CIP^(R) 0.12 0.12 0.12 >8 0.06MMX 6791 N. gonorrhoeae CIP^(R) 0.12 0.12 0.12 >8 0.06 MMX 6792 N.gonorrhoeae CIP^(R) 0.12 0.5 0.25 >8 0.03 MMX 6793 N. gonorrhoeae CTX NS0.06 0.5 0.06 0.03 1 MMX 6757 QC = quality control; Cip^(R) =ciprofloxacin-resistant; CTX NS = ceftriaxone non-susceptible ¹CLSI QCranges shown in parenthesis where applicable

TABLE 94 Minimal Inhibitory Concentration (MIC) Values for MicrobionBismuth Thiol Test Agents and Comparators Against Anaerobes MIC (pg/mL)Isolate Type MB-1-B3 MB-2B MB-6 Clindamycin Metronidazole Fidaxomicin B.fragilis QC 2 8 1 1 0.5 >64 MMX 123 (0.5-2)¹   (0.25-1)   (ATCC 25285)C. difficile QC 4 8 4 4 0.5 0.25 MMX 4381 CC^(I) (2-8) (0.12-0.5)(0.06-0.25) (ATCC 700057) C. difficile ribotype 012 4 8 2 >64 0.5 0.5MMX 5681 CC^(R) (NCTC 13307) C. difficile ribotype 027 1 2 2 4 >64 4 MMX5680 CC^(I) (NCTC 13336) MET^(R) C. difficile ribotype 255 4 8 2 4 0.50.5 MMX 8272 CC^(I) C. difficile ribotype 005 4 16 2 >64 0.5 0.5 MMX8279 CC^(R) C. difficile ribotype 010 2 8 2 >64 8 0.5 MMX 8281 CC^(R) QC= quality control; CC^(I) = Clindamycin intermediate resistance; CC^(R)= Clindamycin-resistant; MET^(R) = Metronidazole-resistant ¹CLSI QCranges shown in parenthesis where applicable

TABLE 95 Minimal Inhibitory Concentration (MIC) Values for MicrobionBismuth Thiol Test Agents and Comparators Against Candida species MIC¹(μg/mL) Isolate Type MB-1-B3 MB-2B MB-6 Fluconazole Amphotericin B C.parapsilosis QC 0.5, 1   0.5, 1  0.5, 1  2,2  0.5, 1 MMX 2323 (0.5-4,1-4)²   (0.25-2, 0.5-4) (ATCC 22019) C. parapsilosis FLU^(R) 0.5, 0.5 0.5, 0.5 0.25, 0.5  32, 32 0.5, 1 MMX 7370 C. parapsilosis FLU^(R) 0.5,1   0.25, 1   0.25, 0.5  32, 64  0.5, 0.5 MMX 7555 C. albicans Sensitive4, 16 2, 2 2, 2 0.5, 0.5  0.5, 0.5 MMX 7039 C. albicans Sensitive 2, 161, 4 2, 2 0.25, 0.5   0.25, 0.5 MMX 7055 C. glabrata FLU^(R) 0.5, 1  0.5, 1  0.5, 1  32, 64 0.5, 1 MMX 7086 C. glabrata FLU^(R) 0.5, 1  0.25, 1   0.25, 0.5  >64, >64 0.5, 1 MMX 7318 C. tropicalis FLU^(R) 4,16 2, 4 4, 4  64, >64 0.5, 1 MMX 7247 C. tropicalis FLU^(R) 16, 32  4, 44, 8  32, >64 0.5, 1 MMX 7248 C. tropicalis FLU^(R) 2, 16 1, 2 2, 2 64, >64  0.5, 0.5 MMX 7360 QC = quality control; FLU^(R) =fluconazole-resistant ¹MIC reported after incubation at 24 and 48 hr²CLSI QC ranges shown in parenthesis where applicable

REFERENCES

-   1.) Centers for Disease Control and Prevention. Antibiotic    resistance threats in the United States, 2013. Available from    http://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf.    Accessed on Jun. 13, 2016.-   2.) Boucher H W, Talbot G H, Bradley J S, Edwards J E, Gilbert D,    Rice L B, Scheld M, Spellberg B, Bartlett J. Bad bugs, no drugs: no    ESKAPE! An update from the Infectious Diseases Society of America.    Clin Infect Dis 2009; 48: 1-12.-   3.) Rice L B. Federal funding for the study of antimicrobial    resistance in nosocomial pathogens: no ESKAPE. J Infect Dis 2008;    197: 1079-1081.-   4.) Clinical and Laboratory Standards Institute (CLSI). Methods for    Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow    Aerobically; Approved Standard—Tenth Edition. Clinical and    Laboratory Standards Institute document M07-A10. CLSI, 940 West    Valley Road, Suite 1400, Wayne, Pa. 19087-1898 USA, 2015.-   5.) CLSI. Performance Standards for Antimicrobial Susceptibility    Testing; Twenty-Sixth Informational Supplement. CLSI document    M100-S26. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pa. 19087    USA, 2016.-   6.) CLSI. Methods for Antimicrobial Susceptibility Testing of    Anaerobic Bacteria; Approved Standard—Eighth Edition. CLSI document    M11-A8. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pa.    19087-1898 USA, 2012.-   7.) CLSI. Reference Method for Broth Dilution Antifungal    Susceptibility Testing of Yeasts; Approved Standard—Third Edition.    CLSI document M27-A3. CLSI, 940 West Valley Road, Suite 1400, Wayne,    Pa. 19087-1898 USA, 2008.-   8.) CLSI. Reference Method for Broth Dilution Antifungal    Susceptibility Testing of Yeasts; Fourth Informational Supplement.    CLSI document M27-S4. CLSI, 940 West Valley Road, Suite 1400, Wayne,    Pa. 19087-1898 USA, 2012.

Example 12 Susceptibility Testing of Three Bismuth Thiols AgainstVancomycin-Intermediate S. aureus and β-lactamase ProducingGram-Negative Bacteria Introduction

The in vitro activity of BisEDT and two additional bismuth-thiolinvestigational agents (MB-2B and MB-6) was determined for isolates ofEscherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa andAcinetobacter baumannii characterized for extended-spectrum β-lactamases(ESBL) and/or carbapenem resistance. In addition,vancomycin-intermediate Staphylococcus aureus (VISA) were evaluated. Themajority of the isolates tested in the current study weremultidrug-resistant (MDR) as defined by resistance to at least threedifferent antibiotic classes. Susceptibility to the investigationalcompounds and relevant comparators was determined by broth microdilutionconducted in accordance with guidelines from the Clinical and LaboratoryStandards Institute (CLSI; 2,3).

Materials and Methods

Test Compounds: The test agents BisEDT (MB-1-B3; Lot No. ED268-1-11-01),MB-2B, and MB-6 were stored at room temperature, in the dark, untilassayed. The solvent and diluent for the test agents was DMSO (Sigma;St. Louis, Mo.; Lot No. SHBB9319V) and the prepared stock concentrationwas 6,464 μg/mL (101× the final test concentration).

Comparator drugs were supplied and shown in Table 96 below:

TABLE 96 Comparator drugs Test Agents Supplier Catalog Number Lot NumberSolvent/Diluent BisEDT Microbion — ED268-1-11-01 DMSO/DMSO MB-2BMicrobion — TA-8-167-01 DMSO/DMSO MB-6 Microbion — 5-21-14 DMSO/DMSOAmikacin Sigma A2324-5G 058K0803 H₂O/H₂O Ceftazidime Sigma C3809-1G076M4770V H₂O/H₂O Clavulanate Sigma 33454-100MG STBH5214 Phos. buff. pH6.0 Clindamycin Sigma C5269-100MG 021M1533 H₂O/H₂O Daptomycin Cubist —MCB2009 H₂O/H₂O Levofloxacin Sigma 28266-1G-F BCBF7004V H₂O + NaOH/H₂OLinezolid Selleck Chemicals S1408 S140802 H₂O/H₂O Meropenem USP 1392454J0K434 H₂O/H₂O Vancomycin Sigma V2002-1G 080M1341V H₂O/H₂O

Test compounds were evaluated at a concentration range of 0.06-64 μg/mL.For Gram-negative test isolates, amikacin and ceftazidime (alone andwith clavulanate at a fixed concentration of 4 μg/mL) were evaluatedover a concentration range of 0.06-64 μg/mL; meropenem and levofloxacinwere evaluated over a concentration range of 0.008-8 μg/mL. For thetesting of S. aureus, clindamycin, daptomycin, levofloxacin andvancomycin were evaluated at a concentration range of 0.008-8 μg/mL;linezolid was tested from 0.03-32 μg/mL.

Organisms: The test organisms as shown in Tables 97-101 consisted ofclinical isolates from the Micromyx (MMX) repository and referencestrains from the American Type Culture Collection (ATCC; Manassas, Va.),National Collection of Type Cultures (NCTC; Public Health England,Salisbury, UK), the Network on Antimicrobial Resistance inStaphylococcus aureus (NARSA; BEI Resources, Manassas, Va.), and theCenters for Disease Control and Prevention (CDC; Atlanta, Ga.). The testorganisms were maintained frozen at −80° C. Prior to testing, theisolates were cultured on Tryptic Soy Agar with 5% sheep blood (BAP;Becton Dickson [BD]/BBL; Sparks, Md.; Lot Nos. 9080650 and 9108563) at35° C. Relevant ATCC quality control (QC) organisms (Table 102) wereincluded during testing in accordance with CLSI guidelines (3). Furtherdetails on the genetic characterization of the isolates where availablecan be found in Table 103.

Media: Cation-adjusted Mueller Hinton broth (CAMHB; BD; Lot No. 8190586)was used as the medium for testing (2, 3). For testing daptomycin,calcium was supplemented with 25 mg/mL Ca²⁺, resulting in a finalconcentration of 50 mg/mL Ca²⁺ (2, 3).

MIC Assay Procedure: MIC values were determined using a brothmicrodilution procedure described by CLSI (2, 3). Automated liquidhandlers (Multidrop 384, Labsystems, Helsinki, Finland; Biomek 2000 andBiomek FX, Beckman Coulter, Fullerton Calif.) were used to conductserial dilutions and liquid transfers.

To prepare the drug mother plates, which would provide the serial drugdilutions for the replicate daughter plates, the wells of columns 2through 12 of standard 96-well microdilution plates (Costar 3795) werefilled with 150 μl of the appropriate diluent for each row of drug. Thetest articles and comparator compounds (300 μl at 101× the highestconcentration to be tested) were dispensed into the appropriate wells incolumn 1. The Biomek 2000 was then used to make 2-fold serial dilutionsin the mother plates from column 1 through column 11. The wells ofcolumn 12 contained no drug and served as the organism growth controlwells for the assay.

The daughter plates were loaded with 190 μL per well of RPMI using theMultidrop 384. The test panels were prepared on the Biomek FX instrumentwhich transferred 2 μL of drug solution from each well of a mother plateto the corresponding well of each daughter plate in a single step.

A standardized inoculum of each test organism was prepared per CLSImethods to equal a 0.5 McFarland standard, followed by a dilution of1:20. The plates were then inoculated with 10 μL of the diluted inoculumusing the Biomek 2000 from low to high drug concentration resulting in afinal concentration of approximately 5×10⁵ CFU/mL.

Plates were stacked 3-4 high, covered with a lid on the top plate,placed into plastic bags, and incubated at 35° C. for 16 to 20 hr(vancomycin was read for S. aureus after 24 hr incubation time). The MICwas recorded as the lowest concentration of drug that inhibited visiblegrowth of the organism.

Results and Discussion

As shown in Table 102, results for BisEDT and comparators were withinCLSI established QC ranges against the relevant ATCC QC isolates, thusvalidating the susceptibility testing conducted during the study.

The activity of BisEDT, MB-2B, and MB-6, against the resistantGram-negative bacilli are shown by species in Tables 97-100. BisEDTmaintained potent activity with MIC values of 0.5-2 μg/mL acrossisolates with the exception of one isolate of P. aeruginosa (CDC 241)which had an MIC of 4 μg/mL (Table 99) and several isolates of K.pneumoniae with MIC values of 4-8 μg/mL (Table 98). The activity ofBisEDT was not impacted by β-lactamase production or resistance toaminoglycosides (amikacin MIC≥64 μg/mL), fluoroquinolones (levofloxacinMIC≥2, 4, and 8≥64 for Enterobacteriaceae, P. aeruginosa, and A.baumannii, respectively).

Overall, MB-6 had MIC values that were either identical or within 2-foldof those observed with BisEDT; exceptions included select K. pneumoniaewhere MB-6 MIC values were lower than those of BisEDT. The activity ofBisEDT and MB-6 was greater than that of MB-2B, particularly for P.aeruginosa and A. baumannii. The MIC values observed with BisEDT, MB-2B,and MB-6 against Gram-negative bacilli were comparable to those observedin prior studies (1, 4).

The activity of BisEDT, MB-2B, and MB-6 against VISA is shown in Table101. BisEDT had potent MIC values of ≤0.06-0.25 μg/mL against theseisolates. As with Gram-negative bacilli, the activity of BisEDT wascomparable to that observed with MB-6 and was greater than that observedwith MB-2B. Of note, two of the VISA isolates (NRS 13 and 27) from NARSAhad vancomycin MIC values of 2 μg/mL, which indicated that duringtesting in this study they tested as vancomycin-susceptible. The othertwo isolates with vancomycin MIC values in the susceptible range (NRS 2and 24) are heterogenous VISA (hVISA) for which vancomycin MIC valuesare known to vary. Resistance to levofloxacin and clindamycin (MICvalues≥4 μg/mL) was observed with all isolates except NRS 13 and did notimpact BisEDT activity. Two of the isolates were also non-susceptible todaptomycin (NRS 13 and 22); all were susceptible to linezolid (MICvalues≤4 μg/mL). The activity observed with BisEDT in this study wascomparable to that observed previously (1, 4).

In summary, BisEDT showed potent activity against geneticallycharacterized β-lactam-resistant Gram-negative bacilli, the majority ofwhich were MDR, and reference isolates of VISA. The activity of BisEDTwas not impacted by resistance to β-lactams or any other class evaluatedin this study. Finally, the activity of BisEDT and MB-6 was comparableagainst the evaluated bacteria and exceeded that observed with MB-2B.

TABLE 97 Activity of BisEDT, MB-2B, MB-6 and comparators againstEscherichia coli MIC (μg/mL) Isolate β-lactamase Type BisEDT MB-2B MB-6CAZ CAZ/CLAV MEM LVX AMK ATCC 35218 ESBL 1 2 1 0.12 ≤0.06/4 ≤0.008≤0.008 1 MMX 5755 ESBL 1 4 2 >64    1/4 0.015 8 16 MMX 5756 ESBL 1 21 >64  0.5/4 ≤0.008 4 64 MMX 5758 ESBL 1 4 1 16  0.25/4 ≤0.008 8 1 CDC451 KPC 1 2 1 64   16/4 1 >8 4 MMX 5745 KPC 1 4 2 32   16/4 2 8 0.5 CDC114 ESBL/KPC 2 2 2 >64   64/4 2 4 1 ATCC BAA-2471 NDM 1 2 2 >64  >64/4 >8 >8 64 CDC 435 NDM 1 2 1 >64   >64/4 >8 >8 >64 CDC 503ESBL/NDM 1 2 2 >64   >64/4 8 >8 >64 CDC 118 ESBL/NDM 1 4 1 >64   >64/40.06 8 >64 ATCC = American Type Culture Collection, MMX = Micromyx, CDC= Centers for Disease Control and Prevention, ESBL = extended-spectrumβ-lactamase, KPC = K. pneumoniae carbapenemase, NDM = New Delhimetallo-β-lactamase, CAZ = ceftazidime, CLAV = clavulanate, MEM =meropenem, LVX = levofloxacin, AMK = amikacin

TABLE 98 Activity of BisEDT, MB-2B, MB-6 and comparators againstKlebsiella pneumoniae MIC (μg/mL) Isolate β-lactamase Type BisEDT MB-2BMB-6 CAZ CAZ/CLAV MEM LVX AMK MMX 9029 ESBL 4 2 2 4 0.12/4  0.015 0.120.5 CDC 112 KPC 2 4 2 >64 >64/4 >8 4 16 ATCC BAA-1705 ESBL/KPC 2 41 >64 >64/4 8 >8 16 CDC 113 ESBL/KPC 8 32 1 >64 >64/4 >8 4 16 CDC 115ESBL/KPC 8 16 2 >64 >64/4 >8 >8 0.5 CDC 120 ESBL/KPC 2 4 2 >64 64/4 >8 >8 16 CDC 126 ESBL/KPC 8 32 2 4 8 4 0.015 2 CDC 129 ESBL/KPC 24 4 >64 >64/4 8 >8 32 CDC 135 ESBL/VIM 4 8 4 >64 >64/4 1 >8 16 CDC 138ESBL/NDM 4 16 2 >64 >64/4 >8 >8 >64 CDC 158 ESBL/NDM/OXA 2 42 >64 >64/4 >8 4 2 ATCC = American Type Culture Collection, MMX =Micromyx, CDC = Centers for Disease Control and Prevention, ESBL =extended-spectrum β-lactamase, KPC = K. pneumoniae carbapenemase, NDM =New Delhi metallo-β-lactamase, VIM = metallo-β-lactamase, OXA = class Dcarbapenemases, CAZ = ceftazidime, CLAV = clavulanate, MEM = meropenem,LVX = levofloxacin, AMK = amikacin

TABLE 99 Activity of BisEDT, MB-2B, MB-6 and comparators againstPseudomonas aeruginosa β-lactamase MIC (μg/mL) Isolate Type BisEDT MB-2BMB-6 CAZ CAZ/CLAV MEM LVX AMK CDC 356 KPC 1 4 1 64 64/4 >8 0.12 2 CDC439 IMP 2 4 1 >64 >64/4  >8 8 >64 CDC 444 VIM 1 4 1 32 32/4 >8 8 >64 CDC457 VIM 1 32 2 >64 64/4 >8 >8 2 CDC 231 KPC/OXA 2 16 2 >64 >64/4  >8 >88 CDC 230 VIM/OXA 0.5 4 2 64 32/4 >8 8 >64 CDC 241 IMP/OXA 4 328 >64 >64/4  >8 8 32 CDC 246 NDM/OXA 2 8 2 >64 >64/4  >8 >8 >64 CDC 250NDM/OXA 2 8 2 >64 >64/4  >8 >8 >64 CDC 516 KPC/AmpC 1 4 1 64 64/4 >80.25 2 CDC 518 KPC/AmpC 1 4 1 32 32/4 >8 >8 16 CDC = Centers for DiseaseControl and Prevention, KPC = K. pneumoniae carbapenemase, NDM = NewDelhi metallo-β-lactamase, IMP = metallo-β-lactamase, VIM =metallo-β-lactamase, OXA = class D carbapenemases, AmpC = class Ccephalosporinase, CAZ = ceftazidime, CLAV = clavulanate, MEM =meropenem, LVX = levofloxacin, AMK = amikacin

TABLE 100 Activity of BisEDT, MB-2B, MB-6 and comparators againstAcinetobacter baumannii β-lactamase MIC (μg/mL) Isolate Type BisEDTMB-2B MB-6 CAZ CAZ/CLAV MEM LVX AMK NCTC 13304 OXA 0.5 >160.5 >64 >64/4 >8 1 0.5 CDC 307 OXA 1 32 0.5 64  64/4 8 8 >64 CDC 311 OXA1 32 0.5 >64 >64/4 >8 2 >64 CDC 312 OXA 1 32 0.5 >64 >64/4 4 2 1 CDC 273ESBL/OXA 1 32 0.5 64  64/4 >8 >8 >64 CDC 274 ESBL/OXA 1 320.5 >64 >64/4 >8 8 16 CDC 275 ESBL/OXA 0.5 32 0.5 >64 >64/4 >8 4 >64 CDC277 ESBL/OXA 1 >16 1 >64 >64/4 >8 8 16 CDC 284 ESBL/OXA 1 >16 1 64 16/4 >8 8 32 CDC 308 ESBL/OXA 1 32 0.5 64  64/4 8 4 >64 CDC 313ESBL/OXA 1 32 0.5 >64 >64/4 >8 2 4 NCTC = National Collection of TypeCultures, CDC = Centers for Disease Control and Prevention, ESBL =extended-spectrum β-lactamase, OXA = class D carbapenemases, CAZ =ceftazidime, CLAV = clavulanate, MEM = meropenem, LVX = levofloxacin,AMK = amikacin

TABLE 101 Activity of BisEDT, MB-2B, MB-6 and comparators againstvancomycin-intermediate Staphylococcus aureus MIC (μg/mL) Isolate BisEDTMB-2B MB-6 VAN DAP CLI LVX LZD NRS 1 (hVISA) 0.12 1 0.12 4 1 >8 4 1 NRS2 ≤0.06 0.5 ≤0.06 0.5 0.25 >8 4 4 NRS 3 0.25 2 0.25 8 1 >8 >8 1 NRS 220.25 2 0.25 4 2 >8 8 1 NRS 4 0.25 1 0.25 4 0.5 >8 4 1 NRS 13 0.12 1 0.122 2 0.06 0.12 2 NRS 18 0.25 1 0.25 4 0.5 >8 4 1 NRS 24 (hVISA) 0.25 1 12 0.5 >8 >8 2 NRS 27 0.25 1 0.25 2 0.25 >8 8 2 NRS = Network onAntimicrobial Resistance in Staphylococcus aureus, MMX = Micromyx, VISA= vancomycin-intermediate S. aureus, hVISA = heterogenousvancomycin-intermediate S. Aureus, VAN = vancomycin, DAP = daptomycin,CLI = clindamycin, LVX = levofloxacin, LZD = linezolid

TABLE 102 Activity of Bis-EDT, MB-2B, MB-6 and comparators againstrelevant ATCC QC organisms MIC (μg/mL) CAZ + Organism Isolate BisEDTMB-2B MB-6 CAZ CLAV MEM LVX AMK E. coli ATCC 25922 0.5 2 1 0.25  0.25/4≤0.008 ≤0.008 1 (0.5-4)¹ (0.06-0.5) (0.008-0.06) (0.008-0.06) ATCC 352181   2 1 0.12 ≤0.06/4 ≤0.008 ≤0.008 1 (0.008-0.06) K. pneumoniae ATCCBAA-1705 2   4 1 >64     >64/4 8   >8    16 (8-64) P. aeruginosa ATCC27853 1   2 1 2      2/4 0.25 0.5  2 (0.5-4) (1-4) (0.12-1) (0.5-4)(1-4) MIC (μg/mL) Organism Isolate BisEDT MB-2B MB-6 VAN DAP CLI LVX LZDS. aureus ATCC 29213 0.12 1 0.12 0.5 0.5 0.25 0.06 4 (0.12-1) (0.5-2)(0.12-1) (0.06-0.25) (0.06-0.5) (1-4) ATCC = American Type CultureCollection, CAZ = ceftazidime, CLAV = clavulanate, MEM = meropenem, LVX= levofloxacin, AMK = amikacin, VAN = vancomycin, DAP = daptomycin, CLI= clindamycin, LVX = levofloxacin, LZD = linezolid QC ranges inparentheses

TABLE 103 Available genetic characterization data on test isolatesOrganism Isolate Genetic Characterization Information E. coli ATCC 35218TEM-1 BAA-2471 NDM-1 CDC 435 NDM CDC 451 KPC MMX 5745 KPC, TEM, DFR MMX5755 SHV, TEM, OXA-1, AAD, ANT, SUL1, SUL2, GYR MMX 5756 SHV, TEM,OXA-9, AAD, SUL2, GYR MMX 5758 SHV, TEM, CTX-M-1, GYR CDC 503 CTX-M-15,NDM-1, OXA-181 CDC 114 aadB, cmlA1, dfrA5, KPC-3, strA, strB, sul1,sul2, TEM-1B CDC 118 aac(3)-IIa, catA1, CMY-6, dfrA29, NDM-1, OmpF,OXA-2, rmtC, strA, strB, sul1, TEM-1A K. pneumoniae BAA-1705 KPC-2, TEM,SHV MMX 9029 CTX-M1, SHV, TEM, AAC, SUL2 CDC 112 aac(6′), aph(3′),aph(4), catA1, cmlA1, dfrA12, KPC-3, mph(A), oqxA, oqxA, oqxB, sul1,sul3 CDC 113 aac(6′)-Ib, aph(3′)-Ia, aph(4)-Ia, catA1, cmlA1, dfrA12,KPC-3, mph(A), OmpK35, OmpK36, oqxA, oqxA, oqxB, SHV-11, sul1, sul3 CDC115 aph(3′)-Ia, aph(4)-Ia, catA1, cmlA1, dfrA12, KPC-3, mph(A), OmpK35,oqxA, oqxA, oqxB, sul1, sul3, TEM-1A CDC 120 aac(6′)-33, aac(6′)-Ib,aadA2, aadB, aph(3′)-Ia, dfrA12, KPC-2, mph(A), OmpK35, oqxA, oqxA,oqxB, sul1, sul2, TEM-1D CDC 126 aac(6′)Ib-cr, catB3, dfrA1, fosA,KPC-2, OmpK36, oqxA, oqxA, OXA-1, sul1, TEM-1B CDC 129 aac(6′)-Ib,aadA2, aph(3′)-Ia, catA1, dfrA12, KPC-3, mph(A), OmpK35, oqxA, oqxA,oqxB, sul1, TEM-1A CDC 135 aac(3)-IIa, aac(6′)-Ib, aph(3′)-XV, catB2,dfrA14, OmpK35, oqxA, oqxA, OXA-9, SHV-12, sul1, TEM-1A, tet(D), VIM-1CDC 138 aadA2, ARR-3, CTX-M-15, dfrA12, dfrA14, mph(A), NDM-7, oqxA,oqxA, SHV-11, strA, strB, sul1, sul2, TEM-1B CDC 158 aac(3)-IId,aac(6′)Ib-cr, CTX-M-15, dfrA14, dfrA30, fosA, NDM-1, oqxA, oqxA, oqxB,OXA-1, strA, strB, sul2, TEM-1B, tet(B) P. aeruginosa CDC 439 IMP CDC444 VIM CDC 457 VIM CDC 356 KPC CDC 230 aac(3)-Id, aadA2, cmlA1, clfrB5,OXA-4, OXA-50, PAO, tet(G), VIM-2 CDC 231 aac(6′)-IIc, KPC-5, OXA-2,OXA-50, PAO CDC 241 aac(6′)-IIc, aadA7, catB7, IMP-1, OXA-101, OXA-50,OXA-9, PAO, sul1 CDC 246 aadB, NDM-1, OXA-10, OXA-50, PAO, rmtD2,tet(G), VEB-1 CDC 250 aadB, NDM-1, OXA-10, OXA-50, PAO, rmtD2, tet(G),VEB-1 CDC 516 PDC-101; KPC-2 CDC 518 PDC-103; KPC-2 A. baumannii NCTC13304 OXA-27 CDC 273 aac(3)-IIa, ADC-25, aph(3′)-Ic, aph(3′)-VIa,OXA-23, OXA-66, strA, strB, sul2 CDC 274 aac(3)-Ia, ADC-25, aph(3′)-Ic,OXA-66, OXA-72, strA, strB, sul1, sul2, TEM-1D CDC 275 ADC-25,aph(3′)-Ic, armA, mph(E), msr(E), OXA-23, OXA-66, strA, strB, sul2,TEM-1D CDC 277 aac(3)-IIa, OXA-24, OXA-65, strA, strB, sul2, TEM-1B CDC284 aac(3)-IIa, OXA-24, OXA-65, strA, strB, sul2, TEM-1B CDC 307 ADC-25,aph(3′)-Ic, armA, catB8, mph(E), msr(E), OXA-23, OXA- 66, strA, strB,sul1, sul2 CDC 308 ADC-25, armA, catB8, mph(E), msr(E), OXA-71, strA,strB, sul1, TEM-1D CDC 311 ADC-25, aph(3′)-Ic, armA, catB8, mph(E),msr(E), OXA-23, OXA- 82, strA, strB, sul1 CDC 312 aph(3′)-Ic, catA1,OXA-69, sul2, tet(B) CDC 313 aac(3)-Ia, aph(3′)-Ic, catA1, OXA-23,OXA-69, TEM-1D, tet(A)

REFERENCES

-   1.) Beckman E, Wolfe C, Pillar C. In vitro Activity of Bismuth    Thiols and Comparators Against Drug Resistant Gram-positive and    -negative Bacteria and Yeast. Final Report Aug. 24,    2016-Microbion 22. Micromyx, Kalamazoo, Mich. 2016.-   2.) Clinical and Laboratory Standards Institute (CLSI). Methods for    Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow    Aerobically; 11^(th) ed. CLSI standard M07. CLSI, 950 West Valley    Road, Suite 2500, Wayne, Pa. 19087 USA, 2018.-   3.) CLSI. Performance Standards for Antimicrobial Susceptibility    Testing; 29^(th) ed. CLSI supplement M100. CLSI, 950 West Valley    Road, Suite 2500, Wayne, Pa. 19087 USA, 2019.-   4.) Schaadt R D, Peterson M, Sweeney D. In vitro Activity of    Bismuth-1,2-ethanedithiol (BisEDT) Against Multiple Clinical    Isolates of Gram-positive and -negative Bacteria. Final Report    08-08-2008-Microbion 2. Micromyx, Kalamazoo, Mich. 2008.

Example 13 Susceptibility Testing of Three Test Compounds Against Yeast(Candida spp.) and Mold (Aspergillus spp.) Introduction

The in vitro activity of BisEDT and two additional bismuth-thiolinvestigational agents (MB-2B and MB-6) was determined for Candida spp.(C. albicans, C. glabrata, C. krusei, and C. auris) and Aspergillus spp.(A. niger, A. terreus, A. fumigatus and A. flavus). Comparators includedamphotericin B, fluconazole, voriconazole, caspofungin and micafungin.Susceptibility was determined by broth microdilution conducted inaccordance with guidelines from the Clinical and Laboratory StandardsInstitute (CLSI; 2,3).

Material and Methods

Test Compounds: The test agents BisEDT (MB-1-B3; Lot No. ED268-1-11-01),MB-2B, and MB-6 were shipped and stored at room temperature, in thedark, until assayed. The solvent and diluent for the test agents wasDMSO (Sigma; St. Louis, Mo.; Lot No. SHBB9319V) and the prepared stockconcentration was 6,464 μg/mL (101× the final test concentration foryeast and fungi). Comparator drugs are shown in Table 104 below:

TABLE 104 Comparator drugs Working Stock Comparator Concentration DrugSupplier Lot No. Solvent/Diluent (μg/mL) Amphotericin Sigma 086M4012VDMSO 6464 B Fluconazole USP H1l308 DMSO 6464 Caspofungin Sigma 086M4750VDMSO 808 Micafungin Astellas 023070 DMSO 808 Voriconazole USP R032E0DMSO 808

Microbion test compounds, amphotericin B, and tluconazole were evaluatedover a concentration range of 0.06-64 μg/mL for yeast and mold.Caspofungin, micafungin, and voriconazole were tested from 0.008-8μg/mL.

Organisms: The test organisms consisted of clinical isolates from theMicromyx (MMX) repository and reference strains from the American TypeCulture Collection (ATCC, Manassas, Va.). The test organisms weremaintained frozen at −80° C. Prior to testing, yeast were sub-culturedon Sabouraud Dextrose Agar (Becton, Dickson and Company; Sparks, Md.;Lot Nos. 9032625, 9074672) at 35° C. The molds were obtained fromenumerated fungal stocks previously prepared at Micromyx and stored in a0.1% TWEEN® saline solution, at 4° C., until use. C. krusei ATCC 6258,C. parapsilosis ATCC 22019, A. fumigatus ATCC MYA-3626, and A. flavusATCC 204304 were included for purposes of quality control (4, 5).

Media: The medium employed for the testing of the yeast and moldisolates was RPMI 1640 from Hyclone Laboratories (Logan, UT; Lot No.AC10257966A) buffered with MOPS from EMD Millipore (Burlington, Mass.;Lot No. 3173588) (2, 3).

MIC Assay Procedures: MIC values were determined using a brothmicrodilution procedure described by CLSI (2, 3). Automated liquidhandlers (Multidrop 384, Labsystems, Helsinki, Finland; Biomek 2000 andBiomek FX, Beckman Coulter, Fullerton Calif.) were used to conductserial dilutions and liquid transfers.

To prepare the drug mother plates, which would provide the serial drugdilutions for the replicate daughter plates, the wells of columns 2through 12 of standard 96-well microdilution plates (Costar 3795) werefilled with 150 μl of DMSO for each row of drug. The test articles andcomparator compounds (300 μl at 101× the highest concentration to betested) were dispensed into the appropriate wells in column 1. TheBiomek 2000 was then used to make 2-fold serial dilutions in the motherplates from column 1 through column 11. The wells of column 12 containedno drug and served as the organism growth control wells for the assay.

The daughter plates were loaded with 190 μL per well of RPMI using theMultidrop 384. The daughter plates were prepared on the Biomek FXinstrument which transferred 2 μL of drug solution from each well of amother plate to the corresponding well of each daughter plate in asingle step.

A standardized inoculum of each organism was prepared per CLSI methods(2, 3). For the yeast, colonies were picked from the streak plate and asuspension was prepared in saline to equal a 0.5 McFarland standard.This suspension was diluted 1:100 in RPMI resulting in a finalconcentration of approximately 0.5−2.5×10³ CFU/mL in the assay. For themolds, based on the previously determined spore count (CFU/mL) from aspore suspension of each Aspergillus spp. isolate, the suspension wasdiluted in RPMI such that a final concentration of approximately0.2−2.5×10⁴ CFU/mL was achieved in the assay. The inoculum for eachorganism was dispensed into sterile reservoirs divided by length(Beckman Coulter), and the Biomek 2000 was used to inoculate the plates.Daughter plates were placed on the Biomek 2000 work surface reversed sothat inoculation took place from low to high drug concentration. TheBiomek 2000 delivered 10 μL of standardized inoculum into each well.DMSO was present at a final concentration of 1% in the test wells.

Plates were stacked four high, covered with a lid on the top plate,placed into plastic bags, and incubated at 35° C. The Candida spp. andAspergillus isolates were read after a 24 hr incubation and again at 48hr.

The microplates were viewed from the bottom using a plate viewer. Foreach mother plate, an un-inoculated solubility control plate wasobserved for evidence of drug precipitation. For yeast, the MICo wasreported as the lowest concentration of drug that completely inhibitedvisible growth of the organism for Microbion test articles, amphotericinB, and voriconazole; the lowest concentration that showed 50% inhibitionrelative to the growth control was reported as the MIC2 for allMicrobion test articles, micafungin, caspofungin, and fluconazole. Formolds, MIC values were reported as the lowest concentration at whichvisible growth was inhibited, and minimum effective concentrations(MECs) were reported for caspofungin, micafungin and the Microbion testagents as the lowest concentration where the growth shifted to a small,rounded, compact hyphal form at the bottom of the well as compared tothe hyphal growth seen throughout the medium in the growth control well.MECs were only reported where observed.

Results and Discussion

As shown in Table 110, results for BisEDT and comparators were withinCLSI established QC ranges against the relevant ATCC QC isolates withthe exception of amphotericin B at 48 hr against C. krusei ATCC 6258. Inthis instance, amphotericin B was within the QC range at 24 hr for thesame isolate and was within the QC range at 48 hr for C. parapsdosisATCC 22019, A. flavus ATCC 204304, and A. fumigatus MYA-3626. Theseresults validate the susceptibility testing conducted during the study.

The activity of BisEDT, MB-2B, and MB-6, against yeast are shown byspecies in Tables 105-108. BisEDT maintained potent activity with 50%inhibition (MIC₂ values) observed from 0.25 — 1 μg/mL across species and100% inhibition (MIC₀ values) observed from 0.5-2 μg/mL for all speciesexcept C. albicans, where MIC₀ values were 1-8 μg/mL. The activity ofBisEDT was not impacted by resistance to echinocandins or azoleanti-fungal agents and was notably maintained against C. auris for whichmulti-drug resistance and azole resistance is particularly an issue (6).The activity of MB-2B and MB-6 was similar to that of BisEDT, with MICvalues identical or within 2-fold those of BisEDT. The MIC valuesobserved with BisEDT, MB-2B, and MB-6 against yeast were comparable tothose observed in a prior study (1).

The activity of BisEDT, MB-2B, and MB-6 against Aspergillus spp. isshown in Table 109. Overall, BisEDT resulted in complete inhibition at24 hr with MIC₀ values of 2-8 μg/mL for most isolates. However, nocomplete inhibition was observed with A. flavus MMX 7935 (MIC₀ of >64μg/mL), and an MIC₀ of 0.5 μg/mL was observed against A. terreus MMX8229. After 48 hr of incubation, complete inhibition by BisEDT was lesscommonly observed across the tested isolates but in instances where MIC₀values were not evident, MEC values were apparent and these values weretypically consistent with the MIC₀ values reported at 24 hr. As withyeast, the activity of BisEDT was comparable to that observed with MB-2Band MB-6. The activity observed among the comparators against theAspergillus spp. was typically more potent than BisEDT, MB-2B, and MB-6with the exception of fluconazole which, as expected, was inactive.

In summary, BisEDT showed potent activity against both Candida spp.,including the notoriously difficult to treat C. auris, and Aspergillusspp. The activity of BisEDT against yeast was not impacted by azole orechinocandin resistance. Finally, the activity of BisEDT, MB-2B, andMB-6 were comparable against the evaluated yeast and mold.

TABLE 105 Activity of BisEDT, MB-2B, MB-6 and comparators againstCandida albicans Test agent and activity (μg/mL) at 24 hr BisEDT MB-2BMB-6 AMP B FLU VORI CASP MICA Isolate No. Type MIC₂ MIC₀ MIC₂ MIC₀ MIC₂MIC₀ MIC₀ MIC₂ MIC₂ MIC₂ MIC₂ ATCC 90028 Susceptible 0.5 4 0.5 2 0.5 20.25 0.25 ≤0.008 0.06 0.03 ATCC 204276 MICA-R 0.5 1 0.5 1 1^(a)  1 0.250.25 ≤0.008 0.25 4 ATCC MYA-2732 FLU-R, VORI-I 1 2 1 2 1   2 0.25 16 0.50.06 0.03 MMX 7067 FLU-R, VORI-R 1 2 0.5 1 0.5 1 0.25 >64 >8 0.06 0.03MMX 7424 CASP-R, MICA-R 1 4 1 2 1   2 0.25 0.5 ≤0.008 8 4 MMX 7053Susceptible 0.5 1 0.5 1 0.5 1 0.12 0.12 ≤0.008 0.06 0.06 MMX 7445 FLU-R,VORI-R 1 8 1 2 1   2 0.12 >64 >8 0.06^(a) 0.03 MMX 7430 Susceptible 1 80.5 1  0.25 0.5 0.25 0.5 ≤0.008 0.06 0.03 MMX 7437 FLU-I 1 8 1 2 1   20.25 4 0.12 0.06 0.03 MMX 7403 Susceptible 0.5 2 0.5 2  0.25 2 0.25 1≤0.008 0.06 0.03 MIC₂ = 50% inhibition, MIC₀ = complete inhibition, AMPB = amphotericin B, FLU = fluconazole, VORI = voriconazole, CASP =caspofungin, MICA = micafungin, I = intermediate, R = resistant^(a)insufficient growth at 24 hr to determine 50% inhibitory endpoint,result after 48 hr incubation reported *there are no AMP B CLSIbreakpoints for yeast (4)

TABLE 106 Activity of BisEDT, MB-2B, MB-6 and comparators againstCandida glabrata Test agent and activity (μg/mL) BisEDT MB-2B MB-6 AMP BFLU VORI CASP MICA Isolate No. Type MIC₂ MIC₀ MIC₂ MIC₀ MIC₂ MIC₀ MIC₀MIC₂ MIC₂ MIC₂ MIC₂ ATCC 90030 Susceptible 0.5 1 0.5 1 0.5 1 0.25 8 0.120.12 0.06 ATCC MYA-2950 Susceptible 0.5 1 0.5 1 0.5 1 0.12 8 0.12 0.120.03 MMX 7103 CASP-R, MICA-R 0.5 1 0.5 1 0.5 1 0.25 4 0.12 >8 8 MMX 7307CASP-R, MICA-R, 0.5 1 0.5 1 0.5 1 0.25 >64 4 >8 8 FLU-R MMX 7285 CASP-R,MICA-R 0.25 0.5 0.25 0.5 0.25 0.5 0.25 32 1 0.5 0.5 MMX 7093 FLU-R 0.5 10.5 1 1 2 0.25 64 2 0.12 0.06 MMX 7101 FLU-R 0.5 1 0.5 1 0.5 1 0.25 64 20.12 0.06 MMX 7087 Susceptible 0.5 1 0.5 1 0.5 1 0.12 1 0.03 0.12 0.03MMX 7549 Susceptible 0.5^(a) 0.5 0.5 1 0.5 1 0.25 4 0.06 0.06 0.06 MMX7111 Susceptible 0.5 1 0.5 1 0.5 1 0.25 2 0.06 0.12 0.06 MIC₂ = 50%inhibition, MIC₀ = complete inhibition, AMP B = amphotericin B, FLU =fluconazole, VORI = voriconazole, CASP = caspofungin, MICA = micafungin,I = intermediate, R = resistant *there are no AMP B CLSI breakpoints foryeast and there are no VORI breakpoints for C. glabrata (4)

TABLE 107 Activity of BisEDT, MB-2B, MB-6 and comparators againstCandida krusei Test agent and activity (μg/mL) BisEDT MB-2B MB-6 AMP BFLU VORI CASP MICA Isolate No. Type MIC₂ MIC₀ MIC₂ MIC₀ MIC₂ MIC₀ MIC₀MIC₂ MIC₂ MIC₂ MIC₂ ATCC 14243 Susceptible 0.5 1 1 2 0.5 1 0.25 16 0.060.012 0.25 ATCC 6258 MICA-I 0.5 1 1 2 0.5 1 0.5 16 0.12 0.25 0.5 MMX7125 Susceptible 0.5 1 1 2 0.5 1 0.5 32 0.25 0.25 0.25 MMX 7128Susceptible 0.5 1 1 2 1 2 0.5 16 0.12 0.25 0.25 MMX 7141 Susceptible 0.51 1 2 1 2 0.25 16 0.12 0.25 0.25 MMX 7153 MICA-R 0.5 1 1 2 0.5 1 0.5 320.25 0.25 1 MMX 7155 Susceptible 0.5 1 1 2 0.5 1 0.25 32 0.25 0.25 0.25MMX 7563 Susceptible 0.5 1 1 2 0.5 1 0.5 32 0.25 0.12 0.25 ATCC 96685Susceptible 0.25 0.5 0.25 0.5 0.25 0.5 0.5 32 0.5 0.12 0.25 MMX 9878MICA-I, VORI-I 0.5 1 0.5 1 0.5 1 0.5 64 1 0.25 0.5 MIC₂ = 50%inhibition, MIC₀ = complete inhibition, AMP B = amphotericin B, FLU =fluconazole, VORI = voriconazole, CASP = caspofungin, MICA = micafungin,I = intermediate, R = resistant *there are no AMP B CLSI breakpoints foryeast and there are no FLU breakpoints for C. krusei (4)

TABLE 108 Activity of BisEDT, MB-2B, MB-6 and comparators againstCandida auris Test agent and activity (μg/mL) BisEDT MB-2B MB-6 AMP BFLU VORI CASP MICA Isolate No. Type MIC₂ MIC₀ MIC₂ MIC₀ MIC₂ MIC₀ MIC₀MIC₂ MIC₂ MIC₂ MIC₂ MMX 9862 Susceptible  0.5 2  0.5 1   0.5 1 0.25 20.03 0.12 0.12 MMX 9863 FLU-R, VORI-R^(c) 1^(a) 1 1^(a) 1 1 20.12 >64 >8 0.12^(b) 0.5 MMX 9864 FLU-R, VORI-R  0.5 1 1^(a) 1  1^(a) 10.25 >64 2 0.25^(b) 0.5 MMX 9865 FLU-R, VORI-R 1^(a) 1 1^(a) 1 1 20.25 >64 8 0.25^(b) 0.25 MMX 9866 FLU-R, VORI-R 1  2 1^(a) 1 1 2 0.5 >648 0.12^(b) 0.5 MMX 9867 FLU-R, VORI-R 1  2 1  2 1 2 0.5 >64 8 0.12 0.5MMX 9868 FLU-R, VORI-R 1^(b)  1^(a) ND 1 1 4 0.25 >64 >8 0.25 0.25 MMX9869 FLU-R, VORI-R 1^(b) 2 1  2 1 4 1 >64 2 0.25 0.25 MMX 9870 FLU-R,VORI-R 1  2 1  2 1 2 1 >64 4 0.25 0.5 MMX 9871 Susceptible 1^(a) 1 1^(a)1 1 2 1 >64 1 0.25^(b) 0.5 MIC₂ = 50% inhibition, MIC₀ = completeinhibition, AMP B = amphotericin B, FLU = fluconazole, VORI =voriconazole, CASP = caspofungin, MICA = micafungin, Azole-R =azole-resistant, ND = not determined (no MIC₂ was apparent)^(a)insufficient growth at 24 hr to determine 50% inhibitory endpoint,result after 48 hr incubation reported ^(b)eagle effect observed (clearMIC₂ endpoint but regrowth at higher test concentrations potentially dueto inducible resistance or compound precipitation in vitro) ^(c)based onbreakpoints published by Lockhart et al., 2017 (6); CLSI breakpointshave not been established for C. auris

TABLE 109 Activity of BisEDT, MB-2B, MB-6 and comparators againstAspergillus spp. Test agent and activity (μg/mL) BisEDT MB-2B MB-6 AMP BFLU VORI CASP MICA Organism Isolate No. MIC₀ 24/48 hr MIC₀ 24/48 hr MIC₀24/48 hr MIC₀ MIC₂ MIC₀ MEC MEC A. fumigatus ATCC MYA-3626 4/8  4/8 2/4  1 >64 0.5 0.06 0.03 ATCC 204305 8/8^(a) 4/8^(a) 8/4^(a) 1 >64 0.50.25 0.25 MMX 5934 4/8^(a) 8/8^(a) 2/4^(a) 1 >64 0.25 0.12 0.06 MMX 5938−/2^(b)  −/2^(b)  −/1^(b)  1 >64 0.25 0.12^(b) ≤0.008^(b) MMX 59394/8^(a) 4/8^(a) 2/4^(a) 1 >64 2 0.12 0.06 A. flavus ATCC 204304 4/8^(a)4/4^(a) 4/4^(a) 1 >64 2 0.06 0.12 ATCC 22546 2/4  1/2  1/2  1 >64 1 0.06≤0.008 MMX 7935 >64/8^(a)  32/4^(a)  8/8  1 >64 0.5 0.03 ≤0.008 A. nigerATCC 29508 −/2^(b)  −/2^(b)  −/2^(b)  0.25 >64 0.5 0.06^(b) ≤0.008^(b)A. terreus MMX 8229 0.5/1   0.5/1   0.25/1     0.25 >64 0.25 0.12^(b)≤0.008^(b) MIC₀ = complete inhibition, MEC = minimum effectiveconcentration, AMP B = amphotericin B, FLU = fluconazole, VORI =voriconazole, CASP = caspofungin, MICA = micafungin ^(a)result shown at48 hr is the MEC, complete inhibition (MIC₀) not observed at thistimepoint ^(b)result reported for 48 hr only, insufficient growth for 24hr read *there are no CLSI breakpoints for Aspergillus spp. (5)

TABLE 110 Activity of Bis-EDT, MB-2B, MB-6 and comparators againstrelevant ATCC QC organisms QC QC Range MIC MIC Organism Test Agent Range24 hr 48 hr 24 hr 48 hr E. coli BisEDT 0.5-4 —   0.5^(a) — ATCC 25922MB-2B — —   2^(a) — MB-6 — —   0.5^(a) — Amphotericin B — — >64 —Fluconazole — — >64 — Voriconazole — — >64 — Caspofungin — — >64 —Micafungin — — >64 — S. aureus BisEDT 0.12-1 —   0.5^(a) — ATCC 29213MB-2B — —   1^(a) — MB-6 — —   1^(a) — Amphotericin B — — >64 —Fluconazole — — >64 — Voriconazole — — >64 — Caspofungin — — >64 —Micafungin — — >64 — C. parapsilosis BisEDT — — 0.5/1^(b)   1/2^(b) ATCC22019 MB-2B — — 1/2^(b) 1/2^(b) MB-6 — — 1/2^(b) 1/4^(b) Amphotericin B0.25-2 0.5-4    0.5   0.5 Fluconazole  0.5-4   1-4  1 2 Voriconazole  0.015-0.12   0.03-0.25    0.12   0.12 Caspofungin 0.25-1 0.5-4    0.25  0.5 Micafungin  0.5-2 0.5-4    0.5 2 C. krusei ATCC BisEDT — —0.5/1^(b)   1/2^(b) 6258 MB-2B — — 1/2^(b) 1/4^(b) MB-6 — — 0.5/1^(b)  1/4^(b) Amphotericin B  0.5-2   1-4    0.5   0.5 Fluconazole    8-64  16-128  16 32  Voriconazole   0.06-0.5 0.12-1     0.12   0.5Caspofungin 0.12-1 0.25-2     0.25   0.25 Micafungin   0.12-0.5 0.12-0.5    0.5   0.5 A. flavus ATCC BisEDT — —  4  8^(c) 204304 MB-2B— —  4  8^(c) MB-6 — —  4  4^(c) Amphotericin B — 0.5-4    0.5 1Fluconazole — — >64 >64  Voriconazole — 0.5-4    0.12 2 Caspofungin — —   0.12   0.06 (MEC) (MEC) Micafungin — —    0.06    0.008 (MEC) (MEC)A. fumigatus BisEDT — —  4 8 ATCC MB-2B — —  4 8 MYA-3626 MB-6 — —  2 4Amphotericin B — 0.5-4    0.5 1 Fluconazole — — >64 >64  Voriconazole —0.25-1     0.25   0.5 Caspofungin — —    0.06   0.06 (MEC) (MEC)Micafungin — —    0.03    0.008 (MEC) (MEC)

REFERENCES

-   1.) Beckman E, Wolfe C, Pillar C. In vitro Activity of Bismuth    Thiols and Comparators Against Drug Resistant Gram-positive and    -negative Bacteria and Yeast. Final Report Aug. 24,    2016-Microbion 22. Micromyx, Kalamazoo, Mich. 2016.-   2.) Clinical and Laboratory Standards Institute (CLSI). Reference    Method for Broth Dilution Antifungal Susceptibility Testing of    Yeasts. 4^(th) ed. CLSI standard M27. CLSI, 940 West Valley Road,    Suite 1400, Wayne, Pa. 19087-1898 USA, 2017.-   3.) CLSI. Reference Method for Broth Dilution Antifungal    Susceptibility Testing of Filamentous Fungi; Approved Standard.    3^(rd) ed. CLSI standard M38. CLSI, 940 West Valley Road, Suite    1400, Wayne, Pa. 19087-1898 USA, 2017.-   4.) CLSI. Performance Standards for Antifungal Susceptibility    Testing of Yeasts. 1^(st) ed. CLSI supplement M60. CLSI, 940 West    Valley Road, Suite 1400, Wayne, Pa. 19087-1898 USA, 2017.-   5.) CLSI. Performance Standards for Antifungal Susceptibility    Testing of Filamentous Fungi; Approved Standard. 1^(st) ed. CLSI    supplement M61. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pa.    19087-1898 USA, 2017.-   6.) Lockhart S R, Etienne K A, Vallabhaneni S, Farooqi J, Chowdhary    A, Govender N, et al. Simultaneous Emergence of Multidrug-Resistant    Candida auris on 3 Continents Confirmed by Whole-Genome Sequencing    and Epidemiological Analyses. Clin Infect Dis 2017; 64:134-140.

Example 14 Studies on Processing Conditions on BisEDT Particle SizeDistribution

It was observed that careful control of the reaction temperature and therate of 1,2 ethanedithiol addition had pronounced impact on the BisEDTparticle size distribution. Representative syntheses are shown below forBisEDT synthesized at 20° C. with a 1.25 hour addition of 1,2-ethane viasyringe pump and BisEDt synthesized at 15° C. with a 1 hour addition of1,2-ethane via syringe pump. Table 111 below shows that temperatureconditions play a critical role in particle size distribution, withprocessing temperatures in the range of 20-30° C. providing BisEDTparticle size distribution that are both small and uniform in particlesize (such as a D90 below 2 microns).

Representative synthesis of BisEDT at 20° C. with 1.25 hour addition ofthiol via syringe pump, and polypropylene cloth for filtration BisEDTsynthesis was performed on 10-g scale. To a 1-L jacketed reactor wascharged USP water (480 mL, 48 vol), followed by 70% HNO3 (34 mL, 3.4vol). A solution of bismuth subnitrate (10 g, 6.84 mmols) in water (43mL, 4.3 vol) and 70% HNO3 (14 mL, 1.4 vol) was added at 20° C. Thereaction mixture was cooled to 15 ° C. for addition of 95% Ethanol. The95% ethanol (180 mL, 18 vol) was then added slowly. (Ethanol addition isexothermic, temperature reached 22° C.). The temperature was thenadjusted back to 20° C. This was followed by dropwise addition of 1,2ethanedithiol (4.3 mL, 7.5 mmols in 95% ethanol in 94 mL, 9.4 vol) overa period of 1.25 hour with the batch temperature at 20° C. during whichtime it turned into a yellow suspension. The reaction was stirred at 20°C. overnight. The reaction mixture was filtered through polypropylenecloth and washed with 95% ethanol (45 mL, 4.5 vol). The wet cake wascharged back to the reactor and slurried in 95% ethanol (380 mL, 38 vol)for two hours at 20° C. The suspension was then filtered (same cloth)and washed with 95% ethanol (30 mL, 3 vol). The wet cake was againslurried in 95% EtOH (170 mL, 17 vol) at 20° C., filtered (same cloth),and washed with 95% ethanol (30 mL, 3 vol). The wet cake was thenslurried in acetone (170 mL, 17 vol) at 20° C. overnight, followed byfiltration (same cloth) and acetone wash (20 mL, 2 vol). The acetone(170 ml, 17 vol) treatment was repeated on the solids and stirred for 2hours. The suspension was filtered (same cloth) and washed with acetone(30 mL, 3 vol) and died at 45° C. and dried at 45° C. (18 hours) toprovide canary yellow solid (10.81 g 91.0%).

Representative synthesis of BisEDT at 15° C. with 1 hour addition ofthiol via syringe pump, and polypropylene cloth for filtration: Thesynthesis BisEDT was performed on 10-g scale, temperature profile wasstudied with data logger. Ethane dithiol was added at 15° C. over 1 hourvia syringe pump and the filtration was performed using PP filter cloth.To a 1-L jacketed reactor was charged USP water (480 mL, 48 vol) andcooled to 15° C., followed by 70% HNO3 (34 mL, 3.4 vol). A solution ofbismuth subnitrate (10 g, 6.84 mmols) in water (43 mL, 4.3 vol) and 70%HNO3 (14 mL, 1.4 vol) was added at the same temperature. The 95% ethanol(180 mL, 18 vol) was then added slowly. (Ethanol addition is exothermic,temperature reached 22.5° C.). It was then allowed to cool to 15° C.This was followed by dropwise addition of 1,2 ethanedithiol (4.3 mL, 7.5mmols in 95% ethanol in 94 mL, 9.4 vol) over an hour with the batchtemperature at 15° C. The reaction was allowed to stir at 15° C.overnight. The reaction mixture was filtered through polypropylene clothand washed with 95% ethanol (45 mL, 4.5 vol). The wet cake was chargedback to the reactor and slurried in 95% ethanol (380 mL, 38 vol) for twohours at 20° C. The suspension was then filtered (same cloth) and washedwith 95% ethanol (30 mL, 3 vol). The wet cake was again slurried in 95%EtOH (170 mL, 17 vol) at 20° C., filtered (same cloth), and washed with95% ethanol (30 mL, 3 vol). The wet cake was then slurried in acetone(170 mL, 17 vol) at 20° C. overnight, followed by filtration (samecloth) and acetone wash (20 mL, 2 vol). The acetone (170 ml, 17 vol)treatment was repeated on the solids and stirred for 2 hours. Thesuspension was filtered (same cloth) and washed with acetone (30 mL, 3vol) and died at 45° C. and dried at 45° C. (18 hours) to provide canaryyellow solid (10.43 g 87.8%).

TABLE 111 Particle Size Distribution of BisEDT samples Sample D (10) μmD (50) μm D (90) μm D [4,3] μm Conditions 1 0.80 2.4 5.9 2.9 DaltonSynthesis Conditions 2 0.58 1.7 3.9 2.0 Dalton Synthesis Conditions 30.59 1.9 4.5 2.3 30° C., 5 h addition of 1,2-ethane dithiol via additionfunnel 4 0.44 1.48 3.1 1.7 30° C., 4 hour addition of 1,2-ethane dithiolvia syringe pump 5 0.33 0.72 1.6 0.86 20° C., 1 h addition of 1,2-ethanedithiol via addition funnel 6 0.34 0.87 1.8 0.98 20° C., 4 h addition of1,2-ethane dithiol via addition funnel 7 0.39 1.3 1.6 1.4 20° C., 1 houraddition of 1,2-ethane dithiol via syringe pump. Sample slurried inEtOH. Cloth filtration 8 0.36 1.0 1.8 1.0 20° C., 1 hour addition of1,2-ethane dithiol via syringe pump. Sample slurried in MeOH. Clothfiltration 9 0.67 1.0 1.9 1.1 20° C., 1 hour addition of 1,2-ethanedithiol via syringe pump. Sample slurried in Abs. MeOH. Cloth filtration10 0.36 0.88 1.7 0.97 20° C., 1 hour addition of 1,2-ethane dithiol viasyringe pump. Sample slurried in IPA. Cloth filtration 11 0.38 1.2 2.41.4 15° C. 1.5 hour addition of 1,2-ethane dithiol via syringe pump.Cloth filtration 12 0.37 1.2 2.4 1.3 20° C., 1.25 hour addition of1,2-ethane via syringe pump 13 0.36 0.98 2.1 1.2 10° C., 1 h addition of1,2-ethane dithiol via syringe pump 14 0.36 1.0 2.1 1.2 10° C. 1 houraddition of 1,2-ethane dithiol via syringe pump. Cloth filtration 150.32 0.72 1.6 0.86 10° C., 4 hours addition of 1,2-ethane dithiol viasyringe pump. Cloth filtration.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

Equivalents

While specific embodiments of the subject disclosure have beendiscussed, the above specification is illustrative and not restrictive.Many variations of the disclosure will become apparent to those skilledin the art upon review of this specification and the claims below. Thefull scope of the disclosure should be determined by reference to theclaims, along with their full scope of equivalents, and thespecification, along with such variations.

1. An aerosol comprising a plurality of dispersed liquid droplets in agas, said liquid droplets comprising a BT composition comprisingbismuth-1,2-ethanedithiol (BisEDT) suspended therein, wherein the BTcomposition comprises a plurality of BisEDT particles having a D90 ofless than about 2 μm; and wherein at least 70% of the liquid dropletshave a MMAD from about 0.4 μm to about 5 μm as measured by cascadeimpaction or laser time of flight.
 2. The aerosol of claim 1, wherein atleast 80% of the dispersed liquid droplets have a MMAD from about 0.4 μmto about 5 μm.
 3. The aerosol of claim 1, wherein at least 90% of thedispersed liquid droplets have a MMAD from about 0.4 μm to about 5 μm.3. The aerosol of claim 1, wherein at least 70% of the dispersed liquiddroplets have a MMAD from about 0.7 μm to about 4 μm.
 4. The aerosol ofclaim 1, wherein at least 80% of the dispersed liquid droplets have aMMAD from about 0.7 μm to about 4 μm.
 5. The aerosol of claim 1, whereinat least 90% of the dispersed liquid droplets have a MMAD from about 0.7μm to about 4 μm.
 6. The aerosol of claim 1, wherein at least 70% of thedispersed liquid droplets have a MMAD from about 0.9 μm to about 3 μm.7. The aerosol of claim 1, wherein at least 80% of the dispersed liquiddroplets have a MMAD from about 0.9 μm to about 3 μm.
 8. The aerosol ofclaim 1, wherein at least 90% of the dispersed liquid droplets have aMMAD from about 0.9 μm to about 3 μm.
 9. The aerosol of claim 1, whereinthe D90 of said particles is less than or equal to about 1.6 μm.
 10. Theaerosol of claim 1, wherein the D50 of said particles is less than orequal to about 1.0 μm.
 11. The aerosol of claim 1, wherein the dispersedliquid droplets comprise BisEDT at a concentration greater than about0.1 mg/mL, about 0.05% to about 1.0% polysorbate 80, about 0.05 to 40 mMsodium chloride, and optionally about 2 to 20 mM sodium phosphate atabout pH 7.4
 12. The aerosol of claim 1, wherein the dispersed liquiddroplets comprise BisEDT at a concentration greater than about 0.25mg/mL, about 0.5% polysorbate 80, about 10 mM sodium chloride, and about10 mM sodium phosphate at about pH 7.4
 13. The aerosol of claim 1,wherein the BisEDT compound has an average half-life of more than 2 dayswhen deposited to the deep lung region.
 14. The aerosol of claim 1,wherein the BisEDT compound has an average half-life of about 4 dayswhen deposited to the deep lung region.
 15. A pharmaceutical compositioncomprising a bismuth-thiol (BT) composition that comprises BisEDTsuspended therein, wherein the BT composition comprises a plurality ofparticles, wherein the D90 of said particles is less than or equal to1.9
 16. The pharmaceutical composition of claim 15, wherein the D90 ofsaid particles is less than or equal to about 1.6 μm.
 17. Thepharmaceutical composition of claim 15, wherein the D50 of saidparticles is less than or equal to about 1.0 μm.
 18. The pharmaceuticalcomposition of claim 15, wherein the composition does not include aliposome.